Optineurin nucleic acid molecules and uses thereof

ABSTRACT

Promoter sequences of the optineurin gene can be used to diagnose, prognose, and treat glaucoma and related disorders. Methods, kits, and nucleic acids capable of detecting or containing polymorphisms located within the promoter region of the optineurin gene are also provided. The promoter sequences can also be used to generate cells, vectors, and nucleic acids useful in a variety of diagnostic and prognostic methods and kits as well as therapeutic compounds, compositions and methods.

FIELD OF THE INVENTION

[0001] Promoter sequences of the optineurin gene can be used todiagnose, prognose, and treat glaucoma and related disorders. Methods,kits, and nucleic acids capable of detecting or containing polymorphismslocated within the promoter region of the optineurin gene are alsoprovided. The promoter sequences can also be used to generate cells,vectors, and nucleic acids useful in a variety of diagnostic andprognostic methods and kits as well as therapeutic compounds,compositions and methods.

BACKGROUND OF THE INVENTION

[0002] The glaucomas are a group of debilitating eye diseases whichrepresent the leading cause of preventable blindness in the UnitedStates and other developed nations. Approximately 2.47 million people inthe United States and over 67 million people world-wide are estimated tobe affected with glaucoma, and over 100,000 Americans are expected todevelop this condition every year. Quigley and Vitale, Invest.Ophthalmol. Vis. Sci. 38:83 (1997); Quigley, Br. J. Ophthalmol. 80:389(1996). Glaucoma is a progressive optic neuropathy characterized by aparticular pattern of visual field loss and optic nerve head damageresulting from a number of different disorders that affect the eye. Ingeneral, glaucomas are characterized by degeneration of the optic nerve.

[0003] Primary Open Angle Glaucoma (POAG), the most common form ofglaucoma, is characterized by cupping of the optic nerve head, analtered visual field, and an open iridocorneal angle. Approximatelyone-half of patients with POAG have high-tension glaucoma, i.e., theyexhibit an intraocular pressure (IOP) greater than the normal IOP ofabout 22 mm Hg. The increased IOP is caused in part by an alteration ofthe trabecular meshwork (TM), which leads to an obstruction of thenormal ability of aqueous humor to leave its chamber surrounding theiris. Elevated IOP can result in progressive visual loss and blindnessif not treated appropriately and in a timely fashion.

[0004] Because increased IOP is a readily measurable characteristic ofglaucoma, the diagnosis of the disease is largely screened for bymeasuring intraocular pressure (tonometry). Strong, Ophthal. Physiol.Opt. 12:3-7 (1992); Greve et al., Can. J. Ophthamol. 28:201-206 (1993).Unfortunately, because glaucomatous and normal pressure ranges overlap,such methods are of limited value unless multiple readings are obtained.Hitchings, Br. J. Ophthamol. 77:326 (1993); Tuck et al., Ophthal.Physiol. Opt. 13:227-232 (1993); Vaughan et al., In: GeneralOphthamology, Appleton & Lange, Norwalk, Conn., pp. 213-230 (1992);Vernon, Eye 7:134-137 (1993). Patients may also have a differentialsensitivity to optic nerve damage at a given IOP. For these reasons,additional methods, such as direct examination of the optic disk anddetermination of the extent of a patient's visual field loss are oftenconducted to improve the accuracy of diagnosis. Greve et al., Can. J.Ophthamol. 28:201-206 (1993). Moreover, these techniques are of limitedprognostic value.

[0005] Approximately one-third to one-half of patients with POAGconsistently have IOP within the statistically normal range of less than22 mmHg, however. Tielsch et al., JAMA 266:269 (1991); Hitchings, Br. J.Ophthalmol. 76:494 (1992); Grosskreutz and Netland, Int. Ophthalmol.Clin. 34:173 (1994). These patients have been considered to havenormal-tension glaucoma (NTG) (also known as low-tension glaucoma (LTG))and exhibit typical glaucomatous cupping of the optic nerve head andvisual field loss. Hitchings and Anderton, Br. J. Ophthalmol. 67:818(1983). See also Werner, Normal-Tension Glaucoma, in Rich et al., eds.The Glaucomas (2nd ed. 1996): 769-797. NTG has been associated with adisproportionately large amount of cupping, larger than average opticdisks, and higher incidences of acquired pit of the optic nerve andoptic disk hemorrhage, as compared to high-tension glaucoma patients.Id. at page 774. Because IOP is not elevated in NTG, tonometrictechniques are of limited diagnostic and prognostic value, and thedisease is often difficult to diagnose until the visual field issignificantly impaired.

[0006] The present invention relates to a gene known as “optineurin”(for optic neuropathy inducing protein), which is also known variouslyas: tumor necrosis factor-alpha (TNF-alpha) inducible protein (Li etal., Mol. Cell. Biol. 18:1601 (1998)); FIP-2 (for adenovirus E3-15.7Kinteracting protein 2); Huntingtin interacting protein L (Faber et al.,Hum. Mol. Genet. 7:1463 (1998)), NEMO-related protein (Schwambom et al.,J. Biol. Chem. 275:22780 (2000)); transcription factor IRA (TFIIIA)interacting protein (Moreland et al., Nucleic Acids Res. 28:1986(2000)); and RAB8-interacting protein (Hattula and Peranen, Curr. Bio.10:1603 (2000)).

[0007] Optineurin has been reported as being associated with adult-onsetPOAG, and mutations in the coding region have been reported ascorrelated with adult-onset NTG/POAG and an increased risk of glaucoma.Rezaie et al., “Adult-Onset Primary Open Angle Glaucoma Caused byMutations in OPTN”, Science 295:1077-1079 (2002). Direct interaction ofoptineurin with E3-14.7K protein has been reported and it has also beenreported that such interaction utilizes TNF-alpha or FAS-Ligand pathwaysto mediate apoptosis, inflammation or vasoconstriction. Li et al., Mol.Cell. Biol. 18:1601 (1998); Wold, J. Cell. Biochem. 53:329 (1993).Optineurin also is reported to function through interactions with otherproteins in cellular morphogenesis and membrane trafficking (RAB 8),vesicle trafficking (Huntingtin), transcription activation (TFIIIA), andassembly or activation of two kinases. Li et al., Mol. Cell. Biol.18:1601 (1998); Hattula and Peranen, Curr. Bio. 10:1603 (2000); Moritzet al., Mol. Biol. Cell 12:2341 (2001); Moreland et al., Nucleic AcidsRes. 28:1986 (2000); Schwamborn et al., J. Biol. Chem. 275:22780 (2000).

SUMMARY OF THE INVENTION

[0008] The present invention includes and provides an isolated nucleicacid molecule that comprises at least 20 consecutive nucleotides but notmore than 1500 consecutive nucleotides of the sequence of SEQ ID NO: 1.The present invention also includes and provides an isolated nucleicacid molecule comprising a promoter which comprises at least 20consecutive nucleotides but not more than 1500 consecutive nucleotidesof the sequence of SEQ ID NO: 1, the promoter being operably linked to aheterologous nucleic acid sequence. Such heterologous nucleic acidsequences may include, without limitation, coding sequences, toxins, andreporter genes, and also may be capable of being transcribed as anantisense RNA.

[0009] The present invention includes a nucleic acid molecule capable ofdetecting a single nucleotide polymorphism selected from table 1 and anucleic acid molecule capable of detecting a single nucleotidepolymorphism in an optineurin promoter by specifically detecting saidsingle nucleotide polymorphism in the optineurin promoter, where thenucleic acid molecule does not specifically hybridize to a nucleic acidmolecule consisting of SEQ ID NO: 1.

[0010] Host cells comprising such nucleic acid molecules are alsoprovided by the present invention, including, without limitation, hostcells selected from the group consisting of non-human mammalian cells,bacterial cells, and isolated human cells.

[0011] The present invention also provides and includes methods fordiagnosing glaucoma in a sample obtained from a cell or a bodily fluidby detecting a polymorphism in a promoter region of the optineurin gene,comprising the steps of: (A) incubating under conditions permittingnucleic acid hybridization, a marker nucleic acid molecule, the markernucleic acid molecule having a nucleic acid sequence that specificallyhybridizes to a sequence selected from the group consisting of SEQ IDNO: 1 and a complement thereof, and a complementary nucleic acidmolecule obtained from a sample, wherein nucleic acid hybridizationbetween the marker nucleic acid molecule and the complementary nucleicacid molecule permits the detection of said polymorphism; (B) permittinghybridization between the marker nucleic acid molecule and thecomplementary nucleic acid molecule; and (C) detecting the presence ofthe polymorphism, wherein the detection of the polymorphism isdiagnostic of glaucoma.

[0012] Also provided by the present invention are methods for prognosingglaucoma in a sample obtained from a cell or a bodily fluid by detectinga polymorphism in a promoter region of the optineurin gene, comprisingthe steps of: (A) incubating under conditions permitting nucleic acidhybridization, a marker nucleic acid molecule, the marker nucleic acidmolecule having a nucleic acid sequence that specifically hybridizes toa sequence selected from the group consisting of SEQ ID NO: 1 andcomplement thereof, and a complementary nucleic acid molecule obtainedfrom a sample, where nucleic acid hybridization between the markernucleic acid molecule and the complementary nucleic acid moleculepermits the detection of the polymorphism; (B) permitting hybridizationbetween the marker nucleic acid molecule and the complementary nucleicacid molecule; and (C) detecting the presence of the polymorphism, wherethe detection of the polymorphism is prognostic of glaucoma.

[0013] Further provided by the present invention are methods fordiagnosing or prognosing glaucoma in a sample obtained from a cell or abodily fluid by detecting a polymorphism in a promoter region of theoptineurin gene, comprising the steps of: (A) incubating underconditions permitting nucleic acid hybridization, a marker nucleic acidmolecule, the marker nucleic acid molecule having a nucleic acidsequence that specifically hybridizes to a optineurin promoter sequenceor its complement, and a complementary nucleic acid molecule obtainedfrom a sample, where nucleic acid hybridization between the markernucleic acid molecule and the complementary nucleic acid moleculepermits the detection of the polymorphism; (B) permitting hybridizationbetween the marker nucleic acid molecule and the complementary nucleicacid molecule; and (C) detecting the presence of the polymorphism, wherethe detection of the polymorphism is diagnostic or prognostic ofglaucoma.

[0014] The methods of the present invention may be used to detect asingle nucleotide polymorphism, and may further comprise a second markernucleic acid molecule.

[0015] The present invention further provides methods for detecting thepresence or absence of a SNP sequence variation in a sample containingDNA, comprising contacting a labeled nucleic acid capable of detecting asingle nucleotide polymorphism selected from table 1 with the DNA of thesample under hybridization conditions and determining the presence ofhybrid nucleic acid molecules comprising the labeled nucleic acid.

[0016] The present invention additionally includes and provides methodsfor detecting the presence or absence of an optineurin promoter sequencevariation, for determining the presence of increased susceptibility to aglaucoma, or to a progressive ocular hypertensive disorder resulting inloss of visual field in a patient, or the severity or progression ofglaucoma in a patient, and methods for detecting a polymorphismcomprising: obtaining a sample containing human genomic DNA, byproviding a nucleic acid molecule capable of detecting a singlenucleotide polymorphism located with an optineurin promoter, anddetecting the presence or absence of said polymorphism.

[0017] Further, the present invention provides kits containing agents ofthe present invention or kits capable of carrying out a method of thepresent invention including, without limitation, kits for determiningthe presence of increased susceptibility to a glaucoma, or to aprogressive ocular hypertensive disorder resulting in loss of visualfield, or the severity or progression of glaucoma in a patient,comprising a labeled nucleic acid capable of detecting a singlenucleotide polymorphism selected from table 1 and a means for detectinghybridization with the labeled nucleic acid, and instructions for usinga kit and kits for determining the presence of increased susceptibilityto a glaucoma, or to a progressive ocular hypertensive disorderresulting in loss of visual field in a patient, or the severity orprogression of glaucoma in a patient, comprising amplification reactionprimers that direct amplification of a selected nucleic acid regioncontaining the characteristic nucleotide substitution of an optineurinpromoter SNP sequence variant and an enzyme for amplifying the regioncontaining the characteristic nucleotide substitution.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 depicts the genomic structure of optineurin, includingregions which interact with other known proteins, putative functionaldomains, sizes of exons, and position and types of mutations observed.

[0019]FIG. 2 depicts an interaction of optineurin with other proteinsand its potential involvement in alternative pathways of FAS-Ligand(left) and TNF-alpha (right). Interactions are depicted with solidarrows; downstream effects are depicted with open arrows; and a blockingeffect of one protein on another is depicted with arrows ending in acircle.

[0020]FIG. 3 provides a diagrammatic representation of the location ofsingle nucleotide polymorphisms (depicted as an “n” above thepolymorphic nucleotide) and DNA motifs (cis elements) and putativeregulatory regions (depicted by labeled lines beneath the nucleotides ofthe motif or regulatory region) and repeat elements (depicted by dottedlines above the nucleotides of the repeat element) in the optineurinpromoter sequence (SEQ ID NO: 1).

DESCRIPTION OF THE NUCLEIC AND AMINO ACID SEQUENCES

[0021] SEQ ID NO: 1 is a Homo sapiens nucleotide sequence of optineurinpromoter.

[0022] SEQ ID NO: 2 is a Homo sapiens nucleotide sequence of theoptineurin promoter and the optineurin coding region.

[0023] SEQ ID NOs: 3 through 463 are Homo sapiens nucleotide sequencesof DNA motifs, repeat elements, and putative regulatory regionsidentified in the human optineurin promoter.

DEFINITIONS

[0024] The following definitions are provided as an aid to understandingthe detailed description of the present invention.

[0025] The abbreviation “EP” refers to patent applications and patentspublished by the European Patent Office, and the term “WO” refers topatent applications published by the World Intellectual PropertyOrganization. “PNAS” refers to Proc. Natl. Acad. Sci. (U.S.A.).

[0026] “Amino acid” and “amino acids” refer to all naturally occurringL-amino acids. This definition is meant to include norleucine,norvaline, ornithine, homocysteine, and homoserine.

[0027] “Chromosome walking” means a process of extending a genetic mapby successive hybridization steps.

[0028] The phrases “coding sequence,” “structural sequence,” and“structural nucleic acid sequence” refer to a physical structurecomprising an orderly arrangement of nucleic acids. The coding sequence,structural sequence, and structural nucleic acid sequence may becontained within a larger nucleic acid molecule, vector, or the like. Inaddition, the orderly arrangement of nucleic acids in these sequencesmay be depicted in the form of a sequence listing, figure, table,electronic medium, or the like.

[0029] A nucleic acid molecule is said to be the “complement” of anothernucleic acid molecule if they exhibit complete complementarity, i.e.,every nucleotide of one of the molecules is complementary to anucleotide of the other. Two molecules are “minimally complementary” ifthey can hybridize to one another with sufficient stability to remainannealed to one another under at least conventional “low-stringency”conditions. Similarly, the molecules are “complementary” if they canhybridize to one another with sufficient stability to remain annealed toone another under conventional “high-stringency” conditions.Conventional stringency conditions are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Haymes et al.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C. (1985).

[0030] The phrases “DNA sequence,” “nucleic acid sequence,” and “nucleicacid molecule” refer to a physical structure comprising an orderlyarrangement of nucleic acids. The DNA sequence or nucleic acid sequencemay be contained within a larger nucleic acid molecule, vector, or thelike. In addition, the orderly arrangement of nucleic acids in thesesequences may be depicted in the form of a sequence listing, figure,table, electronic medium, or the like. “Nucleic acid” refers todeoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0031] “Exogenous genetic material” is any genetic material, whethernaturally occurring or otherwise, from any source that is capable ofbeing inserted into any organism.

[0032] The term “expression” refers to the transcription of a gene toproduce the corresponding mRNA and translation of this mRNA to producethe corresponding gene product (i.e., a peptide, polypeptide, orprotein). The term “expression of antisense RNA” refers to thetranscription of a DNA to produce a first RNA molecule capable ofhybridizing to a second RNA molecule.

[0033] As used herein, the term “glaucoma” has its art recognizedmeaning, and includes primary glaucomas, secondary glaucomas, juvenileglaucomas, congenital glaucomas, and familial glaucomas, including,without limitation, pigmentary glaucoma, high tension glaucoma, lowtension glaucoma, normal tension glaucoma, and their related diseases. Adisease or condition is said to be related to glaucoma if it possessesor exhibits a symptom of glaucoma, for example, and increasedintraocular pressure resulting from aqueous outflow resistance.

[0034] “Homology” refers to the level of similarity between two or morenucleic acid or amino acid sequences in terms of percent of positionalidentity (i.e., sequence similarity or identity).

[0035] As used herein, a “homolog protein” molecule or fragment thereofis a counterpart protein molecule or fragment thereof in a secondspecies (e.g., human optineurin is a homolog of mouse optineurin). Ahomolog can also be generated by molecular evolution or DNA shufflingtechniques, so that the molecule retains at least one functional orstructure characteristic of the original protein (see, e.g., U.S. Pat.No. 5,811,238).

[0036] The phrase “heterologous” refers to the relationship between twoor more nucleic acid or protein sequences that are derived fromdifferent sources. For example, a promoter is heterologous with respectto a coding sequence if such a combination is not normally found innature. In addition, a particular sequence may be “heterologous” withrespect to a cell or organism into which it is inserted (i.e. does notnaturally occur in that particular cell or organism).

[0037] “Hybridization” refers to the ability of a strand of nucleic acidto join with a complementary strand via base pairing. Hybridizationoccurs when complementary nucleic acid sequences in the two nucleic acidstrands contact one another under appropriate conditions.

[0038] “Isolated” refers to a molecule separated from substantially allother molecules normally associated with it in its native state. Morepreferably an isolated molecule is the predominant species present in apreparation. A isolated molecule may be greater than 60% free,preferably 75% free, more preferably 90% free, and most preferably 95%free from the other molecules (exclusive of solvent) present in thenatural mixture. The term “isolated” is not intended to encompassmolecules present in their native state.

[0039] The phrase “operably linked” refers to the functional spatialarrangement of two or more nucleic acid regions or nucleic acidsequences. For example, a promoter region may be positioned relative toa nucleic acid sequence such that transcription of a nucleic acidsequence is directed by the promoter region. Thus, a promoter region is“operably linked” to the nucleic acid sequence.

[0040] “Polyadenylation signal” or “polyA signal” refers to a nucleicacid sequence located 3′ to a coding region that promotes the additionof adenylate nucleotides to the 3′ end of the mRNA transcribed from thecoding region.

[0041] The term “promoter” or “promoter region” refers to a nucleic acidsequence, usually found upstream (5′) to a coding sequence, that iscapable of directing transcription of a nucleic acid sequence into mRNA.The promoter or promoter region typically provide a recognition site forRNA polymerase and the other factors necessary for proper initiation oftranscription. As contemplated herein, a promoter or promoter regionincludes variations of promoters derived by inserting or deletingregulatory regions, subjecting the promoter to random or site-directedmutagenesis, etc. The activity or strength of a promoter may be measuredin terms of the amounts of RNA it produces, or the amount of proteinaccumulation in a cell or tissue, relative to a promoter whosetranscriptional activity has been previously assessed.

[0042] The term “protein” “polypeptide” or “peptide molecule” includesany molecule that comprises five or more amino acids. Typically, peptidemolecules are shorter than 50 amino acids. It is well known in the artthat proteins may undergo modification, including post-translationalmodifications, such as, but not limited to, disulfide bond formation,glycosylation, phosphorylation, or oligomerization. Thus, as usedherein, the term “protein”, “polypeptide” or “peptide molecule” includesany protein that is modified by any biological or non-biologicalprocess.

[0043] A “protein fragment” is a peptide or polypeptide molecule whoseamino acid sequence comprises a subset of the amino acid sequence ofthat protein. A protein or fragment thereof that comprises one or moreadditional peptide regions not derived from that protein is a “fusion”protein.

[0044] “Recombinant vector” refers to any agent such as a plasmid,cosmid, virus, autonomously replicating sequence, phage, or linearsingle-stranded, circular single-stranded, linear double-stranded, orcircular double-stranded DNA or RNA nucleotide sequence. The recombinantvector may be derived from any source and is capable of genomicintegration or autonomous replication.

[0045] “Regulatory sequence” refers to a nucleotide sequence locatedupstream (5′), within, or downstream (3′) to a coding sequence.Transcription and expression of the coding sequence is typicallyimpacted by the presence or absence of the regulatory sequence.

[0046] An antibody or peptide is said to “specifically bind” to aprotein, polypeptide, or peptide molecule of the invention if suchbinding is not competitively inhibited by the presence of non-relatedmolecules.

[0047] “Substantially homologous” refers to two sequences which are atleast 90% identical in sequence, as measured by the BestFit programdescribed herein (Version 10; Genetics Computer Group, Inc., Universityof Wisconsin Biotechnology Center, Madison, Wis.), using defaultparameters.

[0048] “Transcription” refers to the process of producing an RNA copyfrom a DNA template.

[0049] “Transfection” refers to the introduction of exogenous DNA into arecipient host.

[0050] “Transformation” refers a process by which the genetic materialcarried by a recipient host is altered by stable incorporation ofexogenous DNA. The term “host” refers to cells or organisms.

[0051] “Transgenic” refers to organisms into which exogenous nucleicacid sequences are integrated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] One skilled in the art may refer to general reference texts fordetailed descriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (1995); Sambrook et al.,Molecular Cloning, A Laboratory Manual (2d ed.), Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989); Birren et al., Genome Analysis:A Laboratory Manual, volumes 1 through 4, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1997-1999); Coligan et al., Current Protocols inImmunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols inPharmacology, John Wiley & Sons, N.Y.; Fingl et al., The PharmacologicalBasis of Therapeutics (1975), Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., 18th edition (1990); and Albert andJakobiec, Principles and Practice of Ophthalmology, W. B. SaundersCompany (1994). These texts can, of course, also be referred to inmaking or using an aspect of the invention.

[0053] A. Human optineurin

[0054] In the present invention, a human optineurin promoter has beenidentified. The transcription start site of the optineurin codingsequence was determined, and a 5 kb fragment of genomic sequenceupstream of it was cloned. This fragment was found to contain a promoterresponsible for the transcription of optineurin (SEQ ID NO: 1).

[0055] The present invention provides a number of agents, for example,nucleic acid molecules comprising the optineurin promoter, and nucleicacid molecules comprising key regulatory regions of the optineurinpromoter, and provides uses of such agents. The agents of the inventionwill preferably be “biologically active” with respect to either astructural attribute, such as the capacity of a nucleic acid tohybridize to another nucleic acid molecule, or the ability of a proteinto be bound by an antibody (or to compete with another molecule for suchbinding). Alternatively, such an attribute may be catalytic and thusinvolve the capacity of the agent to mediate a chemical reaction orresponse. The agents will preferably be isolated. The agents of theinvention may also be recombinant.

[0056] It is understood that any of the agents of the invention can beisolated and/or be biologically active and/or recombinant. It is alsounderstood that the agents of the invention may be labeled with reagentsthat facilitate detection of the agent, e.g., fluorescent labels,chemical labels, modified bases, and the like. The agents may be used asdiagnostic or therapeutic compositions useful in the detection,prevention, and treatment of glaucoma.

[0057] In one aspect, the invention relates to nucleic acids comprisingnon-coding regions or promoter regions associated with the optineuringene of mammals. These nucleic acids can be used in identifyingpolymorphisms in the genomes of mammals and humans that predict asusceptibility to glaucomas or diseases related to alterations in IOP. Anumber of diagnostic or prognostic methods and kits can be designed fromthese nucleic acids including, without limitation those set forthherein.

[0058] In one embodiment, the nucleic acids can be used to identify ordetect a single base polymorphism in a genome. In other embodiments, twoor more single base polymorphisms or multiple base polymorphisms can beidentified or detected. The detection of a known polymorphism can be thebasis for diagnostic and prognostic methods and kits of the invention.Various methods of detecting nucleic acids can be used in these methodsand with the kits, including, but not limited to, solutionhybridization, hybridization to microarrays containing immobilizednucleic acids or other immobilized nucleic acids, amplification-basedmethods such as PCR and the like, and an appropriate biosensor apparatuscomprising a nucleic acid or nucleic acid binding reagent.

[0059] In another aspect, the invention relates to specific sequencesand variants or mutants from the promoter or 5′ regulatory region of thehuman optineurin gene and nucleic acids incorporating these sequences,variants or mutants. The nucleic acids can be incorporated into themethods and kits of the invention, or used in expression systems,vectors, and cells to produce a protein or polypeptide of interest, orused in methods to identify or detect regulatory proteins or proteinsthat specifically bind to promoter or regulatory regions of theoptineurin gene.

[0060] In one embodiment of this aspect of the invention, for example,nucleic acids have an optineurin promoter SNP sequence variant,represented by characteristic nucleotides, as shown in Table 1 below. Anucleic acid incorporating such a characteristic nucleotide can be usedto identify and determine individuals at risk for developing glaucoma ora progression from an ocular hypertensive state, and may be associatedwith therapeutic responsiveness. For example, a SNP in the MYOC genepromter has been reported to modify therapeutic response and becorrelated with resistance to treatment. Colomb et al., Clin. Genet.60:220-225 (2001). The identification of changes in IOP can be done byany known means, however, the “Armaly” criteria is preferred (seeArmaly, Arch. Ophthalinol. 70:492 (1963); Armaly, Arch. Ophthalmol.75:32-35 (1966); Kitazawa et al., Arch. Ophthalmol. 99:819-823 (1981);Lewis et al., Amer. J. Ophthalmol. 106:607-612 (1988); Becker et al.,Amer. J. Ophthalmol. 57:543 (1967)). TABLE 1 Single NucleotidePolymorphisms (SNPs) in the Optineurin Promoter Location in SEQ ID NO:1Characteristic Nucleotides 391 a/g 691 a/g 709 a/g 887 t/a 894 a/t 987a/c 1112 t/c 1505 c/cc 1606 g/a 2405 g/t 2606 a/g 3313 g/a 3555 t/tt3625 a/g 3629 c/t 3882 t/tt 3988 c/t 4452 g/a

[0061] Sequence comparisons of the optineurin promoter region identify anumber of DNA motifs (cis elements) and regulatory regions, which arelisted below in Table 2. Selected motifs, regulatory regions, and SNPsare shown in FIG. 3. Table 2 contains data obtained by analyzing theoptineurin promoter sequence (SEQ ID NO: 1) with MatInspector, which isa software tool that locates transcription factor binding sites in DNAsequences (Quandt et al., Nucleic Acid Research 23: 4878 (1995)).MatInspector itself, and a full description of the terminology used inTable 2 (e.g., family, matrix, core similarity, matrix similarity) maybe obtained from Genomatrix Software GmbH (München, Germany orwww.genomatix.de). TABLE 2 NAME OF FAMILY/MATRIX FURTHER INFORMATIONPOSITION STRAND CORE SIM. MATRIX SIM. SEQUENCE SEQ. ID NO: OCTB/TST1.01POU-factor Tst-1/Oct-6 10-24 (−) 1.000 0.877 cagcAATTccacttc 3AP1F/TCF11MAFG.01 TCF11/MafG heterodimers, 14-35 (−) 1.000 0.936atgataTGACccagcaattcca 4 binding to sublcass of AP1 sites GATA/GATA.01GATA binding site 24-34 (−) 0.868 0.944 tGATATgaccc 5 (consensus)EV11/EV11.05 ectopic viral integration 29-39 (−) 1.000 0.830 agttatGATAt6 site 1 encoded factor FKHD/FREAC2.01 Fork head RElated 39-54 (−) 1.0000.891 gaaagtTAAAcagaga 7 Activator-2 IRFF/IRF1.01 interferon regulatory43-55 (−) 0.765 0.852 ggaaagtTAAAca 8 factor 1 MYT1/MYT1.02 MyT1 zincfinger tran- 45-55 (−) 1.000 0.881 ggaAAGTtaaa 9 scription factorinvolved in primary neurogenesis XBBF/M1F1.01 M1BP-1/RFX1 complex 47-64(−) 0.850 0.768 gagttccttgGAAAgtta 10 NFAT/NFAT.01 Nuclear factor of48-59 (−) 1.000 0.951 ccttgGAAAgtt 11 activated T-cells IKRS/IK3.01Ikaros 3, potential 66-78 (+) 1.000 0.847 tcctcGGAAtatt 12 regulator oflymphocyte differentiation OCTP/OCT1P.01 octamer-binding factor 67-81(−) 0.980 0.895 ccaaatATTCcgagg 13 1, POU-specific domain PCAT/CAAT.01cellular and viral CCAAT 79-90 (+) 0.847 0.904 tggaaCCAGtga 14 boxAP1F/AP1.01 AP1 binding site  95-103 (−) 0.917 0.955 tTGATTCAg 15BARB/BARBIE.01 barbiturate-inducible 103-117 (+) 1.000 0.873aactAAAGctgagac 16 element PERO/PPARA.01 PPAR/RXR heterodimers 106-125(+) 1.000 0.713 taaagctgagacAAAGtcca 17 AP1F/NFE2.01 NF-E2p45 109-119(−) 1.000 0.865 ttgtcTCAGct 18 HNF4/HNF4.01 Hepatic nuclear factor113-126 (+) 1.000 0.861 gagaCAAAgtccag 19 4 SMAD/SMAD3/01 Smad 3transcription 121-128 (−) 1.000 0.996 GTCTggac 20 factor involved inTGF-beta signaling RORA/RORA1.01 RAR-related orphan 125-137 (+) 1.0000.945 agaccaaGGTCaa 21 receptor alpha 1 SF1F/SF1.01 SF1 steroidogenicfactor 128-136 (+) 1.000 0.988 ccAAGGtca 22 1 AP4R/TAL1ALPHAE47.01Tal-1alpha/E47 heterodimer 141-156 (+) 1.000 0.888 tagggCAGAtgattca 23AP1F/AP1.01 AP1 binding site 149-157 (−) 0.934 0.960 aTGAATCAt 24PIT1/PIT1.01 Pit1, GHF-1 pituitary 152-161 (+) 0.871 0.872 attcATGCag 25specific pou domain transcription factor MINI/MUSCLE_IN1.03 MuscleInitiator Sequence 157-177 (+) 0.862 0.887 tgcagcgacCACAccagtggc 26HAML/AML1.01 runt-factor AML-1 164-169 (−) 1.000 1.000 tgTGGT 27OZAZG/ROAZ.01 Rat C2H2 Zn finger protein 195-210 (−) 0.750 0.813ctgCAGCaaagggtgt 28 involved in olfactory neuronal differentiationMZF1/MZF1.01 MZF1 214-221 (−) 1.000 0.971 gttGGGGa 29 ETSF/ETS1.01c-Ets-1 binding site 232-246 (+) 1.000 0.928 ccaGGAActggtttc 30RPOA/DTYPEPA.01 PolyA signal of D-type 242-251 (−) 1.000 0.834tCCATgaaac 31 LTRs STAT/STAT.01 signal transducers and 244-252 (+) 1.0000.912 ttcatGGAA 32 activators of tran- scription MYT1/MYT1.01 MyT1 zincfinger tran- 251-262 (−) 0.750 0.756 aaAAATtgtctt 33 scription factorinvolved in primary neurogenesis NFAT/NFAT.01 Nuclear factor of 257-268(−) 1.000 0.978 ccatgGAAAaat 34 activated T-cells SRFF/SRF.03 serumresponsive factor 259-273 (−) 0.819 0.842 aCCATCcatggaaaa 35CLOX/CDPCR3HD.01 cut-like homeodomain 264-273 (+) 0.929 0.936 catgGATGgt36 protein MINI/MUSCLEIINI.03 Muscle Initiator Sequence 270-290 (−)1.000 0.862 ccaccccccCACCcaccacca 37 R.REB/RREB1.01 Ras-responsiveelement 271-284 (−) 1.000 0.813 cCCCAcccaccacc 38 binding protein 1SP1F/SP1.01 stimulating protein 1 SP1, 274-286 (+) 0.819 0.890ggtgGGTGggggg 39 ubiquitous zinc finger transcription factor EGRF/WT1.01Wilms Tumor Suppressor 277-289 (+) 1.000 0.937 gggTGGGggggtg 40RREB/RREB1.01 Ras-responsive element 285-298 (−) 1.000 0.851tCCCAaaaccaccc 41 binding protein 1 SEF1/SEF1.01 SEF1 binding site310-328 (−) 0.809 0.686 tgcctgatgaTCTGAggtg 42 PAX6/PAX6.01 Pax-6 paireddomain 317-337 (+) 0.754 0.752 gatcatcAGGCattagagtct 43 proteinPDX1/PDX1.01 Pdx1 (IDX1/IPFI) 322-340 (−) 1.000 0.784atgagactcTAATgcctga 44 pancreatic and intestinal homeodomain TFAHRR/AHRARNT.01 aryl hydrocarbon 344-359 (−) 1.000 0.937tctaggttgCGTGctt 45 receptor/Arnt heterodimers FKHD/XFD3.01 Xenopus forkhead domain 370-383 (−) 1.000 0.852 attgtcAACAgaac 46 factor 3SORY/SOX9.01 SOX (SRY-related HMG box) 374-387 (+) 1.000 0.906tgttgaCAAlaggg 47 CREB/TAXCREB.01 Tax/CREB complex 383-397 (+) 0.7840.838 tagggtTCACgctcc 48 PAX6/PAX6.01 Pax-6 paired domain 384-404 (+)1.000 0.766 agggttcACGCtcctatgaaa 49 protein E2FF/E2F.03 E2F, involvedin cell 384-396 (−) 0.774 0.773 gagCGTGaaccct 50 cycle regulation,interacts with Rb 107 protein AHRR/AHRARNT.01 aryl hydrocarbon 387-402(−) 1.000 0.900 tcataggagCGTGaac 51 receptor/Amt heterodimersOCT1/OCT1.05 octamer-binding factor 1 402-415 (−) 0.888 0.903ctgcattagATTTt 52 AP4R/AP4.03 activator protein 4 408-425 (+) 1.0000.831 taatgCAGCtgctgatct 53 MYOD/MYF5.01 Myf5 myogenic bHLH protein410-421 (+) 1.000 0.948 atgCAGCtgctg 54 SP1F/GC.01 GC box elements429-442 (+) 1.000 0.903 aagaGGCGgagctt 55 EGRF/WT1.01 Wilms TumorSuppressor 452-464 (−) 1.000 0.892 gggTGGGtgagca 56 VMYB/VMYB.02 v-Myb462-470 (−) 1.000 0.951 agcAACGgg 57 PERO/PPARA.01 PPAR/RXR heterodimers494-513 (+) 0.807 0.695 tcctgagaggccACAGgcca 58 HNF4/HNF4.01 Hepaticnuclear factor 4 501-514 (+) 0.750 0.848 aggcCACAggccag 59 B2TF/E2.01BPV bovine papilloma virus 522-537 (−) 0.852 0.878 aaaccccgggTGGTga 60regulator E2 RREB/RREB1.01 Ras-responsive element 528-541 (−) 1.0000.827 cCCCAaaccccggg 61 binding protein 1 GKLF/GKLF.01 gut-enrichedKrueppel-like 543-556 (−) 0.950 0.916 caataaagcaGGGG 62 factorCLOX/CDP.01 cut-like homeodomain 546-557 (−) 1.000 0.780 ccAATAaagcag 63protein RPOA/LPOLYA.01 Lentiviral Poly A signal 549-556 (−) 1.000 1.000cAATAAAg 64 HOXF/HOX1-30.1 Hox-1.3, vertebrate 550-579 (+) 1.000 0.748tttattggacataATTAttaggtcgtgttc 65 homeobox protein ECAT/NFY.02 nuclearfactor Y 550-560 (−) 1.000 0.914 tgtCCAAtaaa 66 (Y-box binding factor)PCAT/CAAT.01 cellular and viral CCAAT 551-562 (−) 1.000 0.916tatgtCCAAtaa 67 box HMYO/S8.01 S8 555-570 (+) 1.000 0.970tggacataATTAttag 68 NKXH/NKX25.02 homeo domain factor 559-566 (+) 0.9440.950 cATAAtta 69 Nkx-2.5/Csx, tinman homolog low affinity sitesGREF/PRE.01 Progesterone receptor 560-586 (+) 1.000 0.881atattattaggtcgTGTTctttttgg 70 MEF2/MEF2.01 myogenic enhancer factor 2573-588 (−) 0.750 0.742 cacCAAAaagaacacg 71 EBOX/USF.02 upstreamstimulating 618-625 (+) 0.875 0.938 cCACATgc 72 factor CDXF/CD2.01 Cdx-2mammalian caudal 620-638 (−) 1.000 0.900 ggtgaatTTTAtggcatgt 73 relatedintestinal transcr. factor MEF2/AMEF2.01 myocyte eithancer factor623-640 (+) 1.000 0.817 tgccaTAAAattcacccc 74 RPOA/DTYPEPA.01 PolyAsignal of D-type 624-633 (+) 1.000 0.816 gCCATaaaat 75 LTRs TBPF/TATA.02Mammalian C-type LTR TATA 624-633 (+) 0.925 0.941 gcCATAAAAt 76 boxEBOX/SREBP1.02 sterol regulatory element- 632-642 (+) 1.000 0.832atTCACcccat 77 binding protein 1 PIT1/PIT1.01 Pit1, GHF-1 pituitary649-658 (−) 0.820 0.905 aatcATACat 78 specific pou domain transcriptionfactor AP1F/AP1.01 AP1 binding site 653-661 (−) 0.934 0.960 aTGAATCAt 79HMYO/S8.01 S8 662-677 (+) 1.000 0.969 ggctttcaATTAcact 80 OCTB/TST1.01POU-factor Tst-1/Oct-6 665-679 (+) 1.000 0.902 tttcAATTacactta 81NKXH/NKX31.01 prostate-specific 670-682 (−) 1.000 0.892 ttttAAGTgtaat 82homeodomain protein NKX3.1 TBPF/ATATA.01 Avian C-type LTR TATA box675-684 (−) 0.812 0.833 cTTTTTAagt 83 MYT1/MYT1.01 MyT1 zinc fingertran- 679-689 (+) 1.000 0.899 aaaAAGTtgta 84 scription factor involvedin primary neurogenesis CDXF/CDX2.01 Cdx-2 mammalian caudal 680-698 (−)1.000 0.835 tgatggtTTTAcaactttt 85 related intestinal transcr. factorHOXF/HOX1-3.01 Hox-1.3, vertebrate 685-714 (+) 1.000 0.773ttgtaaaaccatcATTAcaattcaaattta 86 homeobox protein PDX1/PDX1.01 Pdx1(IDX1/IPF1) 687-705 (+) 0.782 0.805 gtaaaaccaTCATtacaat 87 pancreaticand intestinal homeodomain TF SORY/SOX5.01 Sox-5 698-705 (+) 1.000 0.862attaCAATtc 88 RPOA/APOLYA.01 Avian C-type LTR PolyA 702-716 (−) 0.8530.713 ACTAAAtttgaattg 89 signal MYT1/MYT1.01 MyT1 zinc finger tran-703-714 (−) 0.750 0.756 taAATTtgaatt 90 scription factor involved inprimary neurogenesis OCT1/OCT1.02 octamer-binding factor 1 718-727 (−)0.755 0.864 gATGGaaata 91 RREB/RREB1.01 Ras-responsive element 731-744(+) 1.000 0.898 cCCCAaaaatcccc 92 binding protein 1 MZF1/MZF1.01 MZF1740-747 (−) 1.000 0.975 cgaGGGGa 93 PCAT/ACAAT.01 Avian C-type LTR CCAAT771-779 (+) 0.825 0.879 ccCCCAAtt 94 box STAT/STAT3.01 signal transducerand 773-793 (+) 0.750 0.735 cccaatTTCAggcaactactg 96 activator oftranscription 3 GF11/GF11.01 growth factor independence 786-809 (−)1.000 0.938 aagacagaAAtcagaccagtagtt 96 1 zinic finger protein acts astranscriptional 1RFF/1SRE.01 interferon-stimulated 814-828 (−) 1.0000.825 cagaaaagGAAAgta 97 response element NFAT/NFAT.01 Nuclear factor of814-825 (−) 1.000 0.953 aaaagGAAAgta 98 activated T-cells SRFF/SRF.02serum response factor 818-831 (−) 0.847 0.895 gtCCAGaaaaggaa 99RPOA/DTYPEPA.01 PolyA signal of D-type 832-841 (−) 0.750 0.797tACATtaaat 100 LTRs OCTP/OCT1P.01 octamer-binding factor 1, 834-848 (−)0.849 0.863 ctccatATACattaa 101 POU-specific domain XSEC/STAF.01 Se-CystRNA gene tran- 862-883 (−) 0.778 0.765 gctaCCCCagatgccaaagact 102scription activating factor LYMF/TH1E47.01 Thing 1/E47 heterodimer,866-881 (+) 1.000 0.914 tttggcatCTGGggta 103 TH1 bHLH member specificexpression in a variety of embryonic tissues HOXF/HOX1-3.01 Hox-1.3,vertebrate 881-910 (+) 1.000 0.783 agcaagtacgaatATTAgtctaccacctca 104homeobox protein OCTP/OCT1P.01 octamer-binding factor 1, 885-899 (−)0.980 0.909 actaatATTCgtact 105 POU-specific domain SEF1/SEF1.01 SEF1binding site 904-922 (−) 0.809 0.684 tttatgtgcaTCTGAggtg 106CDXF/CDX2.01 Cdx-2 mammalian caudal 911-929 (−) 1.000 0.863taatattTTTAtgtgcatc 107 related intestinal transcr. factor OCT1/OCT1.05Octamer-binding factor 1 915-928 (−) 1.000 0.891 aatatttttATGTg 108OCT1/OCT1.05 Octamer-binding factor 1 922-935 (+) 0.944 0.894aaatattacATATc 109 CREB/E4BP4.01 E4P4, bZIP domain, tran- 925-936 (−)1.000 0.878 agatatGTAAta 110 scriptional repressor GATA/GATA.01 GATAbinding site 926-936 (−) 0.868 0.942 agatatGTAAtaat 111 (consensus)VBPF/VBP.01 PAR-type chicken 926-935 (+) 1.000 0.889 aTTACatatc 112vitellogenin promotor-binding protein EV11/EV11.03 ectopic viralintegration 932-946 (−) 0.800 0.927 aGAAAagaaaagata 113 site 1 encodedfactor NFAT/NFAT.01 Nuclear factor of 944-955 (−) 1.000 0.951ggaagGAAAaga 114 activated T-cells ETSF/ETS1.01 c-Ets-1 binding site981-995 (−) 1.000 0.909 gaaGGAAgtagagag 115 YY1F/YY1.01 Yin and Yang 11084-1103 (+) 1.000 0.871 gtggcaCCATcttggctcag 116 MYOF/NF1.01 nuclearfactor 1 1093-1110 (+) 1.000 0.940 tctTGGCtcagcgcaacc 117 XBBF/RFX1.01X-box binding protein RFX1 1095-1111 (+) 1.000 0.880 ttggctcagcGCAAcct118 AP1F/NFE2.01 NF-E2 p45 1095-1105 (+) 1.000 0.865 ttggcTCAGcg 119BRAC/BRACH.01 Brachyury 1145-1168 (+) 0.750 0.693agcctctcaagtAGCTgagattac 120 TTFF/TTF1.01 Thyroid transcription1147-1160 (+) 1.000 0.942 cctctCAAGtagct 121 factor-1 (TTF1) bindingsite AP1F/BEL1.01 Bel-1 similar region 1153-1180 (−) 0.919 0.810tggtgcgtgcctgtaatCTCAGctactt 122 GATA/GATA3.01 GATA binding factor 31160-1169 (+) 0.824 0.906 tgaGATTaca 123 AHRR/AHRARNT.01 arylhydrocarbon 1169-1184 (−) 1.000 0.937 gtagtggtgCGTGcct 124 receptor/Amtheterodimers MEF2/HMEF2.01 myocyte enhancer factor 1189-1204 (−) 1.0000.762 atataaAAATtagcca 125 HNF1/HNF1.02 Hepatic nuclear factor 11190-1206 (+) 0.859 0.755 gGCTAatttttatattt 126 TBPF/TATA.01 cellularand viral TATA 1190-1204 (−) 1.000 0.951 ataTAAAaattagcc 127 boxelements FKHD/XFD2.01 Xenopus fork head domain 1192-1205 (−) 1.000 0.905aataTAAAaattag 128 factor 2 OCT1/OCT1.05 octamer-binding factor 11192-1205 (+) 0.944 0.917 ctaatttttATATt 129 MEF2/RSRFC4.02 related toserum response 1197-1213 (−) 1.000 0.885 ctactaaaAATAtaaaa 130 factor,C4 GATA/LMO2COM.02 complex of Lmo2 bound to 1213-1221 (+) 1.000 0.992gaGATAggg 131 Tal-1, E2A proteins; and GATA-1, half-site 2 AREB/AREB6.04AREB6 (Atplal regulatory 1219-1227 (+) 1.000 0.970 ggGTTTcac 132 elementbinding factor 6) CREB/HLF.01 hepatic leukemia factor 1221-1230 (+)0.770 0.832 GTTTcaccat 133 ARP1/ARP1.01 apolipoprotein AI 1248-1263 (+)0.826 0.842 tgaactCCTGacctca 134 regulatory protein 1 T3RH/T3R.01 vErbA,viral homolog of 1251-1266 (−) 1.000 0.924 gtttgaggtcaggagt 135 thyroidhormone receptor alpha 1 RARF/RAR.01 Retinoic acid receptor, 1252-1261(−) 0.897 0.961 aggTCAGgag 136 member of nuclear receptors RORA/RORA1.01RAR-related orphan 1255-1267 (−) 1.000 0.933 cgtttgaGGTCag 137 receptoralpha 1 CREB/CREBP1CJUN.01 CRE-binding protein 1256-1263 (+) 0.769 0.885tgACCTca 138 1/c-Jun heterodimer LYMF/LYF1.01 LyE-1, enriched in B and T1270-1278 (−) 1.000 0.988 tttGGGAgg 139 lymphocytes HOBO/HOGNESS.01Imperfect Hogness/Goldberg 1277-1308 (−) 0.764 0.922ggcggtggctcacgccTGlAatcccagcactt 140 Box IKRS/JK2.01 Ikaros 2, potential1280-1291 (+) 1.000 0.960 tgctGGGAttac 141 regulator of lymphocytedifferentiation CREB/TAXCREB.01 Tax/CREB complex 1291-1305 (−) 0.7840.806 ggtggcTCACgcctg 142 SP1F/SP1.01 stimulating protein 1 SP1,1300-1312 (−) 1.000 0.881 ccagGGCGgtggc 143 ubiquitous zinc fingertranscription factor FKHD/FREAC2.01 Fork head Related 1312-1327 (−)1.000 0.841 agaaagTAAAgaggcc 144 Activator-2 TBPF/MTATA.01 Muscle TATAbox 1324-1340 (+) 1.000 0.855 ttcttTAAAcccagttc 145 MEF2/MEF2.05 MEF21325-1334 (−) 1.000 0.984 ggttTAAAga 146 XBBF/MIF1.01 MIBP-1/RFX1complex 1345-1362 (+) 0.850 0.764 ggggtgtacgGAAAccta 147 AREB/AREB6.04AREB6 (Atpial regulatory 1353-1361 (−) 1.000 0.974 agGTTTccg 148 elementbinding factor 6) E2FF/E2F.02 E2F, involved in cell 1364-1371 (−) 1.0000.849 gcccGAAA 149 cycle regulation, interacts with Rb p107 proteinLYMF/TH1E47.01 Thing 1/E47 heterodimer, 1375-1390 (+) 1.000 0.928actggggtCTGGagag 150 TH1 Bhlh member specific expression in a variety ofembryonic tissues MZF1/MFZF1.01 MZF1 1387-1394 (+) 1.000 0.986 agaGGGGa151 OCT1/OCT1.02 octamer-binding factor 1 1413-1422 (+) 1.000 0.943cATGCaaaac 152 PAX5/PAX9.01 zebrafish PAX9 binding 1438-1461 (+) 0.9330.774 ggtaCCCAttgaagtaagggccat 153 sites RPOA/DTYPEPA.01 PolyA signal ofD-type 1442-1451 (+) 1.000 0.779 cCCATtgaag 154 LTRs VBPF/VBP.01PAR-type chicken 1446-1455 (−) 1.000 0.862 cTTACttcaa 155 vitellogeninpromoter- binding protein CREB/CREBP1.01 cAMP-responsive element1447-1454 (−) 0.766 0.820 ttACTTca 156 binding protein 1 RPOA/LPOLYA.01Lentiviral Poly A signal 1460-1467 (−) 1.000 0.963 aAATAAAt 157XBBF/RFX1.01 X-box binding protein RFX1 1467-1483 (+) 1.000 0.883tttcagcccaGCAAcat 158 HOXF/HOX1-3.01 Hox-1.3, vertebrate 1487-1516 (+)1.000 0.787 cactgataccctcATTAtcaaatggttctt 159 homeobox proteinGATA/GATA1.03 GATA-binding factor 1 1497-1509 (−) 1.000 0.943atttGATAatgag 160 IKRS/IK3.01 Ikaros 3, potential 1516-1528 (+) 1.0000.840 tctagGGAAcagt 161 regulator of lymphocyte differentiationNFAT/NFAT.01 Nuclear factor of 1534-1545 (−) 1.000 0.970 cattgGAAAcag162 activated T-cells AREB/AREB6.04 AREB6 (Atplal regulatory 1534-1542(+) 1.000 0.991 ctGTTTcca 163 element binding factor 6) ECAT/NFY.02Nuclear factor Y 1537-1547 (+) 1.000 0.917 tttCCAAtgac 164 (Y-boxbinding factor) CBBP/CEBP.02 C/EBP binding site 1570-1587 (−) 0.7690.854 ggactttgGGAACctccc 165 NFKB/CREL.01 c-Rel 1570-1579 (+) 1.0000.940 gggaggTTCC 166 IKRS/1K2.01 Ikaros 2, potential 1573-1584 (−) 1.0000.966 ctttGGGAacct 167 regulator of lymphocyte differentiationXSEC/STAF.01 Se-Cys tRNA gene tran- 1574-1595 (+) 1.000 0.781ggttCCCAaagtccagtaggtg 168 scription activating factor SMAD/SMAD3.01Smad3 transcription factor 1617-1624 (+) 1.000 0.997 GTCTgggt 169involved in TGF-beta signaling CP2F/CP2.01 CP2 1619-1629 (−) 1.000 0.915gcagcacCCAG 170 PAX6/PAX6.01 Pax-6 paired domain 1630-1650 (−) 0.7730.753 aggactcAAGCctcagtccct 171 protein ARP1/ARP1.01 Apolipoprotein AI1643-1658 (+) 1.000 0.829 tgagtcCTTGatgctc 172 regulatory protein 1RPAD/PADS.01 Mammalian C-type LTR Poly 1661-1669 (−) 1.000 0.936gGTGGTctt 173 A downstream element ECAT/NFY.01 Nuclear factor Y1680-1695 (+) 1.000 0.899 tcctcCCAAtctgggg 174 (Y-box binding factor)SRFF/SRF.02 Serum response factor 1682-1695 (−) 0.847 0.868ccCCAGatrgggag 175 SP1F/SP1.01 Stimulating protein 1 SP1, 1691-1703 (+)1.000 0.967 tgggGGCGgggga 176 ubiquitous zinc finger transcriptionfactor EGRE/EGR1.01 Egr-1/Kirox-24/NGFI-A 1694-1705 (+) 0.830 0.813gggcgggGGAGt 177 intermediate-early gene product AP1F/AP1.03 Activatorprotein 1 1699-1709 (−) 1.000 0.935 agTGACtcccc 178 CMYB/CMYB.01 c-Myb,important in 1714-1731 (−) 1.000 0.942 tttcacaacaGTTGgagg 179hematopoesis, cellular equivalent to avian myo- blastosis virus oncogenev-myb VMYB/VMYB.02 v-Myb 1716-1724 (+) 0.819 0.895 tccAACTgt 180CEBP/CEBPB.01 CCAAT/enhancer binding 1721-1734 (+) 0.985 0.942ctgttgtGAAAgcc 181 protein beta MINI/MUSCLE_INI.02 Muscle InitiatorSequence 1733-1753 (+) 1.000 0.853 cctccaccCCACccagctctg 182EBOX/SREBP1.02 Sterol regulatory element- 1734-1744 (+) 0.750 0.838ctCCACcccac 183 binding protein 1 PAX5/PAX9.01 Zebrafish PAX9 binding1736-1759 (−) 0.800 0.862 aagaGCCAgagctgggtggggtgg 184 sites SP1F/GC.01GC box elements 1736-1749 (−) 0.872 0.884 gctgGGTGgggtgg 185NFKB/CREL.01 c-Rel 1752-1761 (+) 1.000 0.909 tggctcTTCC 186 ETSF/GABP.01GABP: GA binding protein 1753-1764 (−) 1.000 0.872 ggaGGAAgagcc 187SEF1/SEF1.01 SEF1 binding site 1761-1779 (+) 0.809 0.777ctccaggacaTCTGGggta 188 AP4R/TALIALPHAE47.01 Tal-1alpha/E47 heterodimer1764-1779 (−) 1.000 0.867 tacccCAGAtgtcctg 189 REOA/POLYA.01 MammalianC-type LTR Poly 1778-1795 (−) 0.822 0.823 cAATACAtccatgatcta 190 Asignal EVI1/EVI1.02 Ectopic viral integration 1814-1824 (+) 1.000 0.837agacAAGAaga 191 site 1 encoded factor CMYB/CMYB.01 c-Myb, important in1836-1853 (+) 1.000 0.936 tctaagagctGTTGccag 192 hematopoesis, cellularequivalent to avian myo- blastosis virus oncogene v-myb XBBF/RFX1.01X-box binding protein RFX1 1844-1860 (−) 1.000 0.922 tggactcctgGCAAcag193 MYOF/NF1.01 Nuclear factor 1 1850-1867 (−) 1.000 0.959cgtTGGCtggactcctgg 194 EGRF/EGR3.01 Early growth response gene 1859-1870(−) 1.000 0.795 gaGCGTtggctg 195 3 product NOLF/OLF1.01 olfactoryneuron-specific 1879-1900 (−) 1.000 0.825 aacgagTCCCtttgggcttcct 196factor AREB/AREB6.04 AREB6 (Atpla1 regulatory 1907-1915 (−) 1.000 0.970ctGTTTgga 197 element binding factor 6) GREF/ARE.01 Androgene receptorbinding 1929-1955 (−) 1.000 0.796 gtttgatgttccttgTGTTccctttcc 198 siteIRFF/IRF2.01 Interferon regulatory 1929-1941 (+) 0.750 0.803ggaaaggGAACac 199 factor 2 LDPS/LDSPOLYA.01 Lentiviral Ply A down-1931-1946 (−) 0.862 0.923 tccTTGTgttcccttt 200 stream elementXBBF/RFX1.02 X-box binding protein RFX1 1933-1950 (+) 0.881 0.904agggaacacaaGGAAcat 201 RPOA/DTYPEPA.01 Poly A signal of D-type 1946-1955(+) 0.750 0.777 aACATcaaac 202 LTRs IKRS/IK1.01 Ikaros 1, potential 977-1989 (−) 1.000 0.918 gtgtGGGAaggtt 203 regulator of lymphocytedifferentiation XSEC//STAF.02 Se-Cys tRNA gene tran-  979-1999 (+) 1.0000.864 ccttCCCAcactgctctacat 204 scription activating factorRPOA/DTYPEPA.01 Poly A signal of D-type 2006-2015 (+) 0.75 0.777aCCACaaaac 205 LTRs HAML/AML1.01 runt-factor AML-1 2006-2011 (−) 1.0001.000 tgTGGT 206 HAML/AML1.01 runt-factor AML-1 2014-2019 (−) 1.0001.000 tgTGGT 207 ECAT/NFY.03 Nuclear factor Y 2019-2032 (+) 0.777 0.847atcaACAAAtcagc 208 (Y-box binding factor) TBPF/ATATA.01 Avian C-type LTRTATA BOX 2046-2055 (+) 0.812 0.824 tTATTTCagt 209 IRFF/IRF1.01interferon regulatory 2047-2059 (−) 1.000 0.879 aaaaactGAAAta 210 factor1 VMYB/VMYB.01 v-Myb 2050-2059 (−) 0.876 0.910 aaaAACTgaa 211PAX6/PAX6.01 Pax-6 paired domain 2053-2073 (+) 0.754 0.751agtttttTCGCtgcatttaga 212 protein E2FF/E2F,02 E2F involved in cell cycle2056-2063 (−) 0.857 0.866 gcgaAAAA 213 regulation, interacts with Rbp107 protein PAX5/PAX9.01 zebrafish PAX9 binding 2079-2102 (+) 0.9330.793 tctaCCCAtggaagtgtcaggaa 214 sites MTF1/MTF-1.01 Metal transcriptonfactor 2087-2101 (−) 1.000 0.873 tcctGCACacttcca 215 1, MRE ETSF/ETS2.01c-Ets-2 binding site 2095-2108 (+) 1.000 0.863 tgcaGGAAgatgga 216ZFIA/ZID.01 zinc finger with inter- 2100-2112 (−) 0.777 0.865tgACTCcatcttc 217 action domain AP1F/AP1F1.01 activator protein 12104-2114 (−) 1.000 0.979 ggTGACtccat 218 VMYB/VMYB.02 v-Myb 2113-2121(+) 1.000 0.912 ccaAACGgg 219 ETSF/ELK1.01 Elk-1 2114-2129 (+) 0.8660.83 caaacgGGATgatcca 220 NFKB/NFKAPPAB.02 NF-kappaB 2118-2129 (+) 0.9290.815 cGGGATgatcca 221 AREB/AREB6.04 AREB6 (Atplal Regulatory 2134-2142(−) 1 0.997 ctGTTTctt 222 element binding factor 6) ZFI1A/ZID.01 zincfinger with inter- 2146-2158 (+) 1 0.889 cgGCTCtaacaca 223 action domainXBBF/RFX1.02 X-box binding protein REX1 2149-2166 (+) 1 0.899ctctaacacaaGCAAcag 224 CMYB/CMYB.01 c-Myb, important in hema- 2157-2174(−) 1 0.916 gtttgttgctGTTGcttg 225 topoesis, cellular equivalent toavian myo- blastosis virus oncogene v.-myb CREB/TAXCREB.02 Tax/CREBcomplex 2205-2219 (−) 0.750 0.741 gaggaaaTACGtctt 226 ETSF/ETS2.01c-Ets-2 binding site 2208-2121 (−) 1.000 0.907 aagaGGAAatacgt 227NFAT/NFAT.01 Nuclear factor of 2210-2221 (−) 1.000 0.962 aagagGAAAtac228 activated T-cells EVI1/EVI1.02 ectopic viral integration 2222-2232(−) 1.000 0/854 tgagAAGAtta 229 site 1 encoded factor OAZF/ROAZ.01 RatC2H2 Zn finger protein 2231-2246 (+) 0.750 0.789 cagCATCcttggtga 230involved in olfactory neuronal differentiation EBOR/DELTAEF1.01 deltaEF12238-2248 (−) 1.000 0.985 cctcACCTaag 231 CREB/CREBP1.01 cAMP-responsiveelement 2239-2246 (−) 0.766 0.801 tcACCTaa 232 binding protein 1HNF4/HNF4.02 Hepatic nuclear factor 4 2253-2267 (+) 0.750 0.776tgggtccAGAGgcct 233 GATA/GATA.01 GATA binding site 2262-2272 (−) 1.0001.000 aGATAAggcct 234 (consensus) CREB/E4BP4.01 E4BP4, bZIP domain,2265-2276 (+) 0.758 0.840 ccttatCTAAaa 235 transcriptional repressorTBPF/ATATA.01 Avian C-type LTR TATA box 2265-2274 (−) 0.834 0.850tTAGATAagg 236 XBBF/MIF1.01 MIBP-1/RFX1 complex 2281-2298 (−) 0.8000.774 acggtgcccaGCCAccca 237 EBOX/USF.02 upstream stimulating 2304-2311(+) 0.875 0.931 aCACATgt 238 factor VBPF/VBP.01 PAR-type chicken2305-2314 (−) 1.000 0.863 aTTACatgtg 239 vitellogenin promoter- bindingprotein IKRS/IK2.01 Ikaros 2, potential 2310-2321 (−) 1.000 0.960tgctGGGAttac 240 regulator of lymphocyte differentiation NRSF/NRSF.01neuron-restrictive 2315-2335 (+) 1.000 0.685 cccAGCActttggaaggccga 241silencer factor TANT/TANTIGEN.01 Major T-antigen binding 2326-2344 (+)0.759 0.872 ggaaggcCGAGgcaggtgg 242 site AREB/AREB6.01 AREB6 (Atplalregulatory 2335-2347 (−) 1.000 0.921 gtccACCTgcct 243 element_bindingfactor 6) MYOD/MYOD.02 myoblast determining 2336-2345 (−) 1.000 0.992tcCACCtgcc 244 factor EBOX/SREBP1.02 sterol regulatory element-2344-2354 (+) 1.000 0.791 gaTCACccgag 245 binding protein 1 RARF/RAR.01Retinoie acid receptor, 2353-2362 (+) 0.897 0.961 aggTCAGgag 246 memberof nuclear receptors CREB/HLF.01 hepatic leukemia factor 2384-2393 (−)0.770 0.857 GTTTcgccat 247 CLOX/CDPCR3HD.01 cut-like homeodomain2394-2403 (−) 0.929 0,941 tattGATGag 248 protein OCT1/OCT1.02octamer-binding factor 2409-2418 (+) 1.000 0.941 aATGCaaaaa 249MYT1/MYT1.01 MyT1 zinc finger tran- 2414-2425 (+) 0.750 0.775aaAAATtagctt 250 scription factor involved in primary neurogenesisHAML/AML1.01 runt-factor AML-1 2428-2433 (+) 1.000 1.000 tgTGGT 251IKRS/IK2.01 Ikaros 2, potential 2445-2456 (−) 1.000 0.967 ggctGGGAttac252 regulator of lymphocyte differentiation AHRR/AHRARNT.02 arylhydrocarbon/Arnt 24875-2493  (−) 0.750 0.772 tgggtttGAGTgttctcc 253heterodimers, fixed core CHOP/CHOP.01 heterodimers of CHOP and 2500-2512(−) 1.000 0.943 cacTGCAatctcc 254 C/EBPalpha OCT1/OCT1.01octamer-binding factor 1 2517-2535 (+) 1.000 0.802 gagatTATGccactgcact255 MEF2/MEF2.01 myogenic enhancer factor 2 2565-2580 (+) 0.750 0.752ctcAAAAaataaaata 256 CDXF/CDX2.01 Cdx-2 mammalian caudal 2571-2589 (−)1.000 0.835 caaaggtTTTAttttattt 257 related intestinal transcr. FactorEVI1/EVI1.03 ectopic viral integration 2571-2581 (+) 0.750 0.788aaataAAATaa 258 site 1 encoded factor RPOA/POLYA.01 Mammalian C-Type LTRPoly 2576-2593 (+) 1.000 0.806 aAATAAAacctttggggc 259 A signalE2FF/E2F.02 E2F, involved in cell 2586-2593 (−) 1.000 0.849 gcccCAAA 260cycle regulation, interacts with Rb p107 protein XSEC/STAF.01 Se-CystRNA gene tran- 2606-2627 (−) 1.000 0.812 aatcCCCAgaattctggactct 261scription activating factor NFKB/NFKAPPAB.02 NF-kappaB 2621-2632 (+)0.929 0.877 gGGGATtttcaa 262 HNF1/HNF1.02 Hepatic nuclear factor 12635-265  (+) 0.859 0.778 gGCTAttcaataaatgg 263 RPOA/LPOLYA.01Lentiviral Poly A signal 2642-2649 (+) 1.000 0.971 cAATAAAt 264TBPF/TATA.01 cellular and viral TATA 2646-2660 (−) 1.000 0.925ataTAAAtcccattt 265 box elements HMTB/MTBF.01 muscle-specific Mt binding2649-2657 (+) 1.000 0.901 tgggATTTa 266 site CREB/HLF.01 hepaticleukemia factor 2659-2668 (−) 1.000 0.869 GTTAtgtgat 267 VBPF/VBP.01PAR-type chicken 2659-2668 (−) 0.830 0.886 gTTATgtgat 268 vitellogeninpromoter- binding protein CREB/CREB.03 cAMP-responsive element 2681-2692(+) 1.000 0.915 tcTGACgcagtt 260 binding protein GATA/GATA1.01 GATAbinding factor 1 2692-2705 (−) 1.000 0.963 tagttGATAggaga 270CLOX/CLOX.01 Clox 2700-2714 (−) 1.000 0.823 aaaATCGaatagttg 271NFAT/NFAT.01 Nuclear factor of 2709-2720 (−) 1.000 0.972 tgaagGAAAatc272 activated T-cells GFI1/GFI1.01 growth factor independence 2728-2751(+) 1.000 0.943 aatttaaaAATCacatcaagggat 273 1 zinc finger protein actsas transcriptional repressor MEF2/MEF2.05 MEF2 2728-2737 (+) 1.000 0.969aattTAAAaa 274 GATA/GATA3.02 GATA-binding factor 3 2746-2755 (+) 0.8120.904 agGGATctaa 275 FKHD/FREAC3.01 Fork head Related 2747-2762 (+)0.750 0.849 gggatCTAAataaaga 276 Activator-3 MEF2/MEF2.05 MEF2 2749-2758(+) 1.000 0.960 gatcTAAAta 277 RPOA/LPOLYA.01 Lentiviral Poly A signal2754-2761 (+) 1.000 0.992 aAATAAAg 278 HMTB/MTBF.01 muscle-specific Mtbinding 2766-2774 (−) 1.000 0.911 agctATTTa 279 site VMYB/VMYB.02 v-Myb2780-2788 (−) 0.819 0.892 cccAACTga 280 SMAD/SMAD3.01 Smad3transcription factor 2788-2795 (+) 1.000 0.993 GTCTggtc 281 involved inTGF beta signaling HNF4/HNF4.02 Hepatic nuclear factor 4 2801-2815 (−)0.750 0.778 aaggaccAAACctct 282 MYT1/MYT1.02 MyT1 zinc finger tran-2815-2825 (−) 1.000 0.897 agaAAGTtcta 283 scription factor involved inprimary neurogenesis HEAT/HSF1.01 heat shock factor 1 2816-2825 (−)1.000 0.98 AGAAagttct 284 MZF1/MZF1.01 MZF1 2847-2854 (−) 1.000 0.978aatGGGGa 285 TBPF/TATA.02 Mammalian C-Type LTR TATA 2852-2861 (−) 0.8850.914 tcTGTAAAAT 286 box GATA/GATA1.03 GATA-binding factor 1 2856-2868(+) 1.000 0.981 tacaGATAaaggg 287 ETSF/PU1.01 Pu. 1 (Pul20) Ets-like2868-2883 (+) 1.000 0.870 gaatgaGGAAgggtaa 288 transcription factoridentified in lymphoid B cells CREB/HLF.01 hepatic leukemia factor2885-2894 (−) 1.000 0.892 GTTActtcat 289 VBPF/VBP.01 PAR-type chicken2885-2894 (−) 1.000 0.913 gTTACttcat 290 vitellogenin promoter- bindingprotein RORA/RORA2.01 RAR-related orphan 2890-2902 (+) 1.000 0.928gtaacttGGTCaa 291 receptor alpha 2 LDPS/LDSPOLYA.01 Lentiviral Poly Adown- 2932-2947 (+) 1.000 0.900 ggaGTGTgtgtgcatg 292 stream elementEBOX/USF.02 upstream stimulating 2943-2950 (−) 0.875 0.933 aCACATgc 293factor NFKB/NFKAPPAB.01 NF-kappaB (p50) 2966-2975 (−) 1.000 0.885GGGGgtgccc 294 MINI/MUSCLE_INI.03 Muscle Initiator Sequence 2967-2987(+) 1.000 0.879 ggcacccccCACCccgacccc 295 REBV/EBVR.01 Epstein-Barrvirus tran- 2967-2987 (−) 1.000 0.828 ggggtcggggtggggGGTGcc 296scription factor R EGRF/WT1.01 Wilms Tumor Suppressor 2968-2980 (−)1.000 0.909 gggTGGGgggtgc 297 SP1F/GC.01 GC box elements 2970-2983 (−)0.872 0.897 tcggGGTGgggggt 298 RREB/RREB1.01 Ras-responsive element2973-2986 (+) 1.000 0.826 cCCCAccccgaccc 299 binding protein 1PCAT/ACAAT.01 Avian C-type LTR CCAAT box 2986-2994 (+) 0.793 0.866ccACCACtg 300 ARP1/ARP1.01 apolipoprotein AI 2993-3008 (−) 1.000 0.861tgattcCTTGctctca 301 regulatory protein 1 MYT1/MYT1.02 MyT1 zinc fingertran- 3015-3025 (−) 1.000 0.893 tcaAAGTtgtt 302 scription factorinvolved in primary neurogenesis IRFF/ISRE.01 interferon-stimulated3033-3047 (+) 1.000 0.800 ctgtaccaGAAActc 303 response elementEGRF/WT1.01 Wilms Tumor Suppressor 3053-3065 (−) 1.000 0.900gtgTGGGaggctc 304 RARF/RAR.01 Retinoic acid receptor, 3085-3094 (−)1.000 0.987 aggTCACcca 305 member of nuclear receptors RORA/RORA1.01RAR-related orphan 3088-3100 (−) 1.000 0.956 agaagaaGGTCac 306 receptoralpha 1 ectopic viral integration site 1 EVI1/EVI1.01 encoded factor3092-3107 (−) 1.000 0.728 agccAAGAgaagaagg 307 OCT1/OCT1.05octamer-binding factor 1 3124-3137 (+) 0.888 0.911 ctcattttaATTCa 308OCTB/TST1.01 POU-factor Tst-1/Oct-6 3125-3139 (−) 1.000 0.961agtgAATTaaaatga 309 RBIT/BRIGHT.01 Bright, B B326 cell 3127-3139 (−)1.000 0.959 agtgaATTAaaat 310 regulator of IgH tran- scriptionNKXH/NKX25.02 homeo domain factor 3129-3136 (+) 1.000 0.874 tTTAAttc 311Nkx-2.5/Csx, tinman homolog low affinity sites GREF/PRE.01 Progesteronereceptor 3140-3166 (+) 1.000 0.847 ttcatagtgttgtttTGTTctcgtttt 312binding site RPOA/POLYA.01 Mammalian C-type LTR Poly 3142-3159 (−) 0.8220.711 gAACAAAacaacactatg 313 A signal AHRR/AHR.01 arylhydrocarbon/dioxin 3193-3210 (−) 0.750 0.840 actccagcttGGGTgaga 314receptor GFI1/GFI1.01 growthfactor independence 3213-3236 (+) 1.0000.953 agtgctgcAATCacagctcattgc 315 1 zinc finger protein acts astranscriptional repressor LYMF/LYF1.01 LyF-1, enriched in B and T3277-3285 (−) 1.000 0.988 tttGGGAgg 316 lymphocytes HOBO/HOGNESS.01Imperfect Hogness/Goldberg 3284-3315 (−) 0.764 0.917cacggtggctcacaccTGTAatcccagcactt 317 Box IKRS/1K2.01 Ikaros 2, potential3287-3298 (+) 1.000 0.960 tgctGGGAttac 318 regulator of lymphocytedifferentiation MYOD/E47.02 TAL1/E47 dimers 3293-3308 (+) 1.000 0.932gattaCAGGtgtgagc 319 AREB/AREB6.02 AREB6 (Atpla1 regulatory 3295-3306(−) 1.000 0.979 tcaCACCtgtaa 320 element binding factor 6) BRAC/TBX5.01T-Box factor 5 site 3297-3308 (+) 1.000 0.991 acaGGTGtgagc 331 (TBX5),mutations related to Holt-Oram syndrome TBPF/MTATA.01 Muscle TATA box3323-3339 (−) 1.000 0.888 ctgttTAAAaccctata 322 FKHD/FREAC2.01 Fork headRelated 3327-3342 (+) 1.000 0.854 gggtttTAAAcagtaa 323 Activator-2MEF2/MEF2.05 MEF2 3329-3338 (+) 1.000 0.986 gtttTAAAca 324 CEBP/CEBP.02C/EBP binding site 3359-3376 (−) 0.957 0.857 tgcctgcgGTAAGtcgta 325NOLF/OLF1.01 olfactory neuron-specific 3383-3404 (−) 1.000 0.822aaagggTCCCcccggggcctgt 326 factor AP2F/AP2.01 activator protein 23388-3399 (−) 0.976 0.895 gtCCCCccgggg 327 MZFl/MZF1.01 MZF1 3391-3398(+) 1.000 0.980 cggGGGGa 328 HEN1/HEN1.01 HEN1 3415-3436 (+) 1.000 0.873ccagggtaCAGCtgtgacaccg 329 AP4R/AP4.01 activator protein 4 3421-3430 (−)1.000 0.974 caCAGCtgta 330 GATA/GATA1.02 GATA-binding factor 1 3448-3461(−) 1.000 0.934 actggGATAatcca 331 NFKB/NFKAPPAB.02 NF-kappaB 3448-3459(−) 0.929 0.822 tGGGATaatcca 1332 FKHD/HFH8.01 HNF-3/Fkh Homolog-83461-3473 (+) 1.000 0.970 tagatAAACaaaa 333 GATA/GATA.01 GATA bindingsite 3462-3472 (+) 1.000 0.949 aGTAAAacaaa 334 (consensus) SORY/SRY.01sex-determining region Y 3464-3475 (+) 1.000 0.946 ataaACAAaaat 335 geneproduct CREB/CREB.02 cAMP-responsive element 3480-3491 (−) 1.000 0.87ggaaTGACgatc 336 binding protein PAX3/PAX3.01 Pax-3 paired domain3482-3494 (+) 1.000 0.785 TCGTcattccatt 337 protein, exressed inembryogenesis, mutations correlate to Waardenburg Syndrome TEAF/TEF1.01TEF-1 related muscle 3484-3495 (+) 1.000 0.834 gtCATTccattt 338 factorPAX1/PAX1.01 Pax1 paired domain 3490-3507 (+) 0.750 0.733CCATttctctctgtatat 339 protein, expressed in the developing vertebralcolumn of mouse embryos NFAT/NFAT.01 Nuclear factor of 3508-3519 (−)1.000 0.966 gcttgGAAAaat 340 activated T-cells BARB/BARBIE.01barbiturate-inducible 3514-3528 (−) 1.000 0.885 atgaAAAGggcttgg 341element OCT1/OCT1.02 octamer-binding factor 1 3520-3529 (−) 0.763 0.823cATGAaaagg 342 AP1F/TCF11MAFG.01 TCF11/MafG heterodimers, 3522-3543 (+)0.777 0.808 ttttcaTGAAtgatcagttatt 343 binding to subclass of AP1 sitesPITI1/PIT1.01 Pit1, GHF-1 pituitary 3527-3536 (−) 1.000 0.855 gatcATTCat344 specific pou domain transcription factor VMYB/VMYB.01 v-Myb3534-3543 (−) 0.876 0.938 aatAACTgat 345 ETSF/ETS2.01 c-Ets-2 bindingsite 3537-3550 (−) 1.000 0.946 tgcaGGAAataact 346 GFI1/GFI1.01 growthfactor independence 3541-3564 (−) 1.000 0.977 aaaaaaaaAATCagtgcaggaaat347 1 zinc finger protein acts as transcriptional repressorAP1F/AP1F1.01 activator protein 1 3592-3602 (−) 1.000 0.968 ggTGACagagt348 EBOX/SREBP1.02 sterol regulatory element- 3617-3627 (−) 0.750 0.791gaTCATgccac 349 binding protein 1 PAX3/PAX3.01 Pax-3 paired domain3628-3640 (+) 0.780 0.765 TCGGctcgctgca 350 protein, expressed inembryogenesis, mutations correlate to Waardenburg Syndrome HEAT/HSF1.01heat shock factor 1 3663-3672 (−) 1.000 0.937 AGAAgaatcg 351XSEC/STAF.02 Se-Gys tRNA gene tran- 3706-3726 (+) 0.810 0.870gagtACCAtcatgcccggcta 352 scription activating factor P53F/P53.01 tumorsuppressor p53 3712-3731 (+) 1.000 0.660 catCATGcccggctaatttt 353MEF2/RSRFC4.02 related to serum response 3729-3745 (−) 1.000 0.885ctactaaaAATAcaaaa 354 factor, C4 SRFF/SRF.01 serum response factor3755-3772 (+) 0.773 0.653 ttcaccaTATTggccagg 355 ECAT/NFY.02 nuclearfactor Y 3760-3770 (−) 1.000 0.920 tggCCAAtatg 356 (Y box bindingfactor) HNF4/HNF4.02 Hepatic nuclear factor 4 3788-3802 (−) 0.750 0.784cagatcgCAAGgtcc 357 LYMF/LYP1.01 LyF-1, enriched in B and T 3813-3821(−) 1.000 0.988 tttGGGAgg 358 lymphocytes HOBO/HOGNESS.01 ImperfectHogness/Godberg 3820-3851 (−) 0.764 0.928cgcggtggctcacgccTGTAatcccagcactt 359 Box IKRS/1K2.01 Ikaros 2, potential3823-3834 (+) 1.000 0.960 tgctGGGAttac 360 regulator of lymphocytedifferentiation CREB/TAXCREB.01 Tax/CREB complex 3834-3848 (−) 0.7840.806 ggtggctCACgcctg 361 EBOX/MYCMAX.03 MYC-MAX binding sites 3848-3857(−) 0.813 0.920 gcCAGGcgcg 362 GATA/GATA3.02 GATA-binding factor 33866-3875 (+) 0.875 0.910 acTGATataa 363 EVI1/EVI1.04 ectopic viralintegration 3868-3882 (+) 1.000 0.809 tGATAtaaaaagaat 364 site 1 encodedfactor MEF2/MEF2.05 MEF2 3869-3878 (+) 1.000 0.968 gataTAAAaa 365TBPF/TATA.01 cellular and viral TATA 3870-3884 (+) 1.000 0.958ataTAAAaagaattt 366 box elements RPOA/APOLYA.01 Avian C-type LTR Poly A3874-3888 (−) 0.829 0.754 AAAAAAattcttttt 367 signal MEF2/MEF2.05 MEF23884-3893 (−) 1.000 0.969 aattTAAAaa 368 EBOX/SREBP1.02 sterolregulatory element- 3899-3909 (+) 0.750 0.849 ttTCTCcccac 369 bindingprotein 1 MZF1/MZF1.01 MZF1 3903-3910 (−) 1.000 1.000 agtGGGGa 370MINI/MUSCLE_INI.03 Muscle Initiator Sequence 3904-3924 (+) 1.000 0.881ccccactccCACCcccaggct 371 RREB/RREB1.01 Ras-responsive element 3904-3917(+) 1.000 0.831 cCCCActcccaccc 372 binding protein 1 EGRF/WT1.01 WilmsTumor Suppressor 3905-3917 (−) 1.000 0.941 gggTGGGagtggg 373 AP2F/AP2.01activator protein 2 3913-3924 (+) 0.976 0.929 caCCCCcaggct 374TBPF/MTATA.01 Muscle TATA box 3919-3945 (+) 1.000 0.917ccttaTAAAgcagcctc 375 HAML/AMLI.01 Runt-factor AML-1 3968-3973 (+) 1.0001.000 tgTGGT 376 ETSF/ELK1.02 Elk-1 3983-3996 (+) 1.000 0.926gggcccGGAAttgg 377 LYMF/THIE47.01 Thing 1/E47 heterodinner, 3991-4006(+) 1.000 0.910 aattgggtCTGGggca 378 TH 1 bHLH member specificexpression in a variety of embryonic tissues PAX5/PAX5.01B-cell-specific activating 4016-4043 (−) 0.904 0.862cccaagAGCAgggcagagaagcaagcaa 379 protein LTUP/TAACC.01 Lentiviral TATAupstream 4037-4059 (−) 1.000 0.838 tgcccctgaggCTAACCccaaga 380 elementPAX5/PAX5.01 B-cell-specific activating 4050-4077 (+) 0.952 0.820ctcaggGGCAgggttgagagtcaggctt 381 protein PCAT/CLTR_CAAT.01 MammalianC-type LTR CCAAT 4056-4080 (−) 0.803 0.758 gcCAAGcctgactctcaaccctgcc 382box MYOD/MYF5.01 Myf5 myogenic bHLH protein 4082-4093 (+) 1.000 0.920aggCAGCaggag 383 ETSF/ELK1.01 Elk-1 4084-4099 (+) 0.800 0.832gcagcaGGAGgtccag 384 SMAD/SMAD3.01 Smad3 transcription factor 4094-4101(−) 1.000 0.996 GTCTggac 385 involved in TOF-beta signalingGATA/GATA2.02 GATA-binding factor 2 4120-4129 (+) 1.000 0.917 ggaGATAcca386 HMTB/MTBF.01 Muscle-specific Mt binding 4121-4129 (−) 0.884 0.912tggtATCTc 387 site EGRF/WT1.01 Wilms Tumor Suppressor 4131-4143 (+)0.813 0.893 gagAGGGcgcatc 388 PERO/PPARA.01 PPAR/RXR heterodimers4143-4162 (−) 1.000 0.694 ctgaaacaggaaAAAGgcag 389 GKLF/GKLF.01gut-enriched Krueppel-like 4146-4159 (−) 0.936 0.918 aaacaggaaaAAGG 390factor NFAT/NFAT.01 Nuclear factor of 4147-4158 (−) 1.000 0.984aacagGAAAaag 391 activated T-cells AREB/AREB6.04 AREB6 (Atpl alregulatory 4154-4162 (+) 1.000 1.000 ctGTTTcag 392 element bindingfactor 6) SORY/SRY.01 sex-determining region Y 4181-4192 (−) 1.000 0.950aaaaACAAaaca 393 gene product FKHD/HFH2.01 HNF-3/Fkh Homolog 2 4183-4194(−) 1.000 0.938 aaaaaAACAaaa 394 EGRF/WT1.01 Wilms Tumor Suppressor4210-4222 (−) 0.813 0.871 gagAGGGagggag 395 EGRF/WT1.01 Wilms TumorSuppressor 4222-4234 (−) 0.813 0.871 gagAGGGagggag 396 GKLF/GKLF.01gut-enriched Krueppel-like 4252-4265 (−) 1.000 0.916 agagagagagAGGG 397factor SP1F/SP1.01 stimulating protein 1 SP1, 4267-4279 (−) 0.844 0.888ggagGGAGgggga 398 ubiquitous zinc finger transcription factorGKLF/GKLF.01 gut-enriched Krueppel-like 4269-4282 (−) 0.950 0.936gaaggagggaGGGG 399 factor OCT1/OCT1.02 octamer-binding factor 14321-4330 (+) 1.000 0.849 gATGCacata 400 EVI1/EVI1.06 ectopic viralintegration 4346-4354 (−) 0.750 0.835 acaAGGTag 401 site 1 encodedfactor TCFF/TCF11.01 TCFl1/KCR-Fl/Nrfl 4353-4365 (+) 1.000 0.991GTCAtcctgctgt 402 homodimers MINI/MUSCLE_INI.01 Muscle InitiatorSequence 4383-4403 (+) 1.000 0.857 tccctcctCCACaccagcaga 403NRSF/NRSF.01 neuron-restrictive 4412-4432 (+) 1.000 0.746ttcAGCAacaagaatagccga 404 silencer factor CLOX/CDPCR3.01 cut-likehomeodomain 4414-4428 (+) 0.888 0.770 cagcaacaagaATAG 405 proteinPCAT/CLTR_CAAT.01 Mammalian C-type LTR CCAAT 4455-4479 (+) 0.803 0.761ccCAAGaagcatcctgcaggctttc 406 box BARB/BARBIE.01 barbiturate-inducible4475-4489 (−) 1.000 0.875 tcaaAAAGcagaaag 407 element MEF2/MMEF2.01myocyte enhancer factor 4489-4504 (−) 1.000 0.892 tgcttTAAAatacact 408TBPF/TATA.02 Mammalian C-type LTR TATA 4494-4503 (−) 0.927 0.938gcTTTAAAAt 409 box TBPF/ATATA.01 Avian C-type LTR TATA box 4520-4529 (+)0.896 0.809 cTATGTAtgc 410 MYT1/MYT1.01 MyT1 zinc finger tran- 4531-4542(−) 0.750 0.776 caTAGTtaactg 411 scription factor involved in primaryneurogenesis GATA/GATA3.02 GATA-binding factor 3 4544-4553 (+) 1.0000.904 ctAGATgtta 412 FKHD/XFD3.01 Xenopus fork head domain 4545-4558 (−)1.000 0.836 aaggttAACAtcta 413 factor MYT1/MYT1.01 MyT1 zinc fingertran- 4548-4559 (−) 0.750 0.775 aaAGGTtaacat 414 scription factorinvolved in primary neurogenesis AP4R/TALIBETA-E47.01 Tal-1 beta/E47heterodimer 4567-4582 (+) 1.000 0.884 aaacaCAGAtggaggc 415 EGRF/EGR1.01Egr-1/Krox-24/NGFI-A 4614-4625 (+) 1.000 0.780 ttctgtgGGCGg 416immediate-early gene product ZFIA/ZID.01 zinc finger with inter-4639-4651 (+) 1.000 0.918 cgGCTCcagcctc 417 action domainCREB/TAXCREB.02 Tax/CREB complex 4657-4671 (+) 1.000 0.700cgggatcTGCGggaa 418 CEBP/CEBP.02 C/EBP binding site 4660-4677 (+) 0.8580.875 gatctgcgGGAAGacacg 419 E2FF/E2F.01 E2F, involved in cell 4662-4676(+) 0.750 0.762 tctgcggGAAGacac 420 cycle regulation, interacts with Rbp107 protein EBOX/NMYC.01 N-Myc 4671-4682 (−) 1.000 0.901 ttcccCGTGtct421 CLOX/CDP.01 cut-like homeodomain 4703-4714 (−) 0.757 0.751tcATTAatcaaa 422 protein HNF1/HNF1.01 hepatic nuclear factor 1 4706-4720(+) 0.775 0.836 gATTAatgatttatt 423 CART/CART1.01 Cart-1 (cartilagehomeo- 4713-4730 (+) 0.791 0.881 gatTTATtttgattaacg 424 protein 1)RPOA/LPOLYA.01 Lentiviral Poly A signal 4714-4721 (−) 1.000 0.963aAATAAAt 425 HNF1/TTNF1.01 hepatic nuclear factor 1 4716-4730 (−) 1.0000.798 cGTTAatcaaaataa 426 COMP/COMP1.01 COMP 1, cooperates with4717-4740 (+) 0.791 0.785 tattttgATTAacgccgtcacagt 427 myogenic proteinsin multicomponent complex CREB/ATF.01 activating transcription 4726-4739(−) 1.000 0.921 ctgTGACggcgtta 428 factor PAX5/PAX5.02 B-cell-specificactivating 4733-4760 (−) 0.842 0.775 agggactgctctaaGGCGtcactgtgac 429protein PAX6/PAX6.01 Pax-6 paired domain 4735-4755 (+) 1.000 0.763cacagtgACGCcttagagcag 430 protein CREB/ATF.01 activating transcription4737-4750 (+) 1.000 0.906 cagTGACgccttag 431 factor WHZF/WHN.01 wingedhelix protein, 4738-4748 (+) 1.000 0.974 agtgACGCctt 432 involved inhair keratinization and thymus epithelium differentiation FKHD/FREAC4.01Fork head RElated 4756-4771 (−) 1.000 0.775 cccgggtgAACAggga 433ACtivator-4 EGRF/NGF1C.01 nerve growth factor- 4795-4806 (+) 0.763 0.835caGCGAgggtgg 434 induced protein C SP1F/SP1.01 stimulating protein 1 SP4812-4824 (+) 1.000 0.895 tgggGGCGgacgc 435 1, ubiquitous zinc fingertranscription factor GKLF/GKLF.01 gut-enriched Krueppel-like 4826-4839(+) 0.950 0.921 ggaaagaggaGGGG 436 factor PCAT/CLTR_CAAT.01 MammalianC-type LTR CCAAT 4827-4851 (−) 0.803 0.780 acCAAGgccccgcccctcctctttc 437box SP1F/SP1.01 stimulating protein 1 SP 4834-4846 (+) 1.000 0.985gaggGGCGgggcc 438 1, ubiquitous zinc finger transcription factorRREB/RREB1.01 Ras-responsive element 4847-4860 (−) 1.000 0.806cCCCAcccgaccaa 439 binding protein 1 TEAF/TEF1.01 TEF-1 related muscle4860-4871 (−) 1.000 0.850 ccCATTccatac 440 factor PAX5/PAX9.01 zebrafishPAX9 binding 4866-4889 (+) 0.866 0.780 aatgGGCAgggtgggggggatggg 441sites RREB/RREB1.01 Ras-responsive element 4868-4881 (−) 1.000 0.795cCCCAccctgccca 442 binding protein 1 EGRF/WT1.01 Wilms Tumor Suppressor4874-4886 (+) 1.000 0.903 gggTGGGggggat 443 RREB/RREB1.01 Ras-responsiveelement 4877-4890 (−) 1.000 0.796 gCCCAtccccccca 444 binding protein 1MZF1/MZF1.01 MZF1 4878-4885 (+) 1.000 0.986 gggGGGGa 445 SP1F/SP1.01stimulating protein 1 SP 4884-4896 (+) 1.000 0.937 gatgGGCGgggta 446 1,ubiquitous zinc finger transcription factor SP1F/SP1.01 stimulatingprotein 1 SP 4900-4912 (+) 1.000 0.961 gatgGGCGgggcc 447 1, ubiquitouszinc finger transcription factor E2FF/E2F.03 E2F, involved in cell4910-4922 (+) 0.806 0.788 gccCGGGaaattc 448 cycle regulation, interactswith RB p107 protein NOLF/OLF1.01 olfactory neuron-specific 4915-4936(+) 1.000 0.843 ggaaatTCCCcggcgcgggcag 449 factor NFKB/NFKAPPAB.01NF-kappaB 4915-4924 (−) 1.000 1 GGGAatttcc 450 IKRS/IK1.01 Ikaros 1,potential 4916-4928 (−) 1.000 0.916 gccgGGGAatttc 451 regulator oflymphocyte differentiation HEN1/HEN1.01 HEN1 4944-4965 (+) 1.000 0.820ctggctgtCAGCtgagccgcgc 452 APAR/AP4.01 activator protein 4 4950-4959 (−)1.000 0.977 ctCAGCtgac 453 SP1F/SP1.01 stimulating protein 1 SP4964-4976 (+) 1.000 0.945 gctgGGCGgggtc 454 1, ubiquitous zinc fingertanscription factor EGRF/NGFIC.01 nerve growth factor- 5018-5029 (−)0.787 0.802 tgGCGGaggggg 455 induced protein C EGRF/NGFIC.01 nervegrowth factor- 5024-5035 (−) 0.787 0.794 cgGCGGtggcgg 456 inducedprotein C EGRF/NGFTC.01 nerve growth factor- 5030-5041 (−) 0.787 0.799ggGCGGcggcgg 457 induced protein C SPIF/SP1.01 stimulating protein 1 SP5032-5044 (−) 1.000 0.898 ggcgGGCGgcggc 458 1, ubiquitous zinc fingertranscription factor AP2F/AP2.01 activator protein 2 5037-5048 (+) 1.0000.957 cgCCCGccggca 459

[0062] As used herein, the term “cis elements capable of binding” refersto the ability of one or more of the described cis elements tospecifically bind an agent. Such binding may be by any chemical,physical or biological interaction between the cis element and theagent, including, but not limited, to any covalent, steric, agostic,electronic and ionic interaction between the cis element and the agent.As used herein, the term “specifically binds” refers to the ability ofthe agent to bind to a specified cis element but not to wholly unrelatednucleic acid sequences. Regulatory region refers to the ability of anucleic acid fragment, region or length to functionally perform abiological activity. The biological activity may be binding to thenucleic or specific DNA sequence. The biological activity may alsomodulate, enhance, inhibit or alter the transcription of a nearby codingregion. The biological activity may be identified by a gel shift assay,in which binding to a nucleic acid fragment can be detected. Othermethods of detecting the biological activity in a nucleic acidregulatory region are known in the art (see Current Protocols inMolecular Biology, for example).

[0063] Human transcription factor activator protein 1 (AP1) is atranscription factor that has been shown to regulate genes which arehighly expressed in transformed cells such as stromelysin, c-fos,α₁-anti-trypsin and collagenase. Gutman and Wasylyk, EMBO J. 9.7:2241-2246 (1990); Martin et al., PNAS 85: 5839-5843 (1988); Jones etal., Genes and Development 2: 267-281 (1988); Faisst and Meyer, NucleicAcid Research 20: 3-26 (1992); Kim et al., Molecular and CellularBiology 10: 1492-1497 (1990); Baumhueter et al., EMBO J. 7: 2485-2493(1988). The AP1 transcription factor has been associated with genes thatare activated by 12-O-tetradecanolyphorbol-13-acetate (TPA). Sequencescorresponding to an upstream motif or cis element capable of binding AP1(SEQ ID NOs: 4, 15, 18, 24, 79, 119, 122, 178, 218, 343, and 348) arelocated in the optineurin promoter (SEQ ID NO: 1) at the respectiveresidues indicated in Table 2. In accordance with certain embodiments ofthe present invention, transcription of optineurin molecules can beeffected by agents capable of altering the biochemical properties orconcentration of AP1 or its homologues, including, but not limited to,the concentration of AP1 or its homologues bound to an upstream motif orcis element. Such agents can be used in the study of glaucomapathogenesis. In another embodiment, such agents can also be used in thestudy of glaucoma prognosis. In another embodiment such agents can beused in the treatment of glaucoma.

[0064] A consensus sequence (GR/PR), recognized by both theglucocorticoid receptor of rat liver and the progesterone receptor fromrabbit uterus, has been reported to be involved in glucocorticoid andprogesterone-dependent gene expression. Von der Ahe et al., Nature 313:706-709 (1985). Sequences corresponding to a GC/PR upstream motif or ciselement (SEQ ID NOs: 70 and 312) are located in the optineurin promoter(SEQ ID NO: 1) at the respective residues indicated in Table 2. Inaccordance with the embodiments of the present invention, transcriptionof optineurin molecules can be effected by agents capable of alteringthe biochemical properties or concentration of glucocorticoid orprogesterone or their homologues, including, but not limited to, theconcentration of glucocorticoid or progesterone or their homologuesbound to an GC/PR upstream motif or cis element. Such agents can be usedin the study of glaucoma pathogenesis. In another embodiment, suchagents can also be used in the study of glaucoma prognosis. In anotherembodiment such agents can be used in the treatment of glaucoma.

[0065] A consensus sequence for a vitellogenin gene-binding protein(VBP) upstream motif or cis element has been characterized. Iyer et al.,Molecular and Cellular Biology 11: 4863-4875 (1991). Expression of theVBP gene commences early in liver ontogeny and is not subject tocircadian control. Sequences corresponding to an upstream motif or ciselement capable of binding VBP (SEQ ID NOs: 112, 155, 239, 268 and 290)are located in the optineurin promoter (SEQ ID NO: 1) at the respectiveresidues indicated in Table 2. In accordance with the embodiments of thepresent invention, transcription of optineurin molecules can be effectedby agents capable of altering the biochemical properties orconcentration of VBP or its homologues, including, but not limited to,the concentration of VBP or its homologues bound to an VBP upstreammotif or cis element. Such agents can be used in the study of glaucomapathogenesis. In another embodiment, such agents can also be used in thestudy of glaucoma prognosis. In another embodiment such agents can beused in the treatment of glaucoma.

[0066] NFkB (or NFKB) is a transcription factor that is reportedlyassociated with a number of biological processes including T-cellactivation and cytokine regulation. Lenardo et al., Cell 58: 227-229(1989). A consensus upstream motif or cis element capable of bindingNFkB has been reported. Sequences corresponding to an upstream motif orcis element capable of binding NFkB (SEQ ID NOs: 166, 186, 221, 262,294, 332 and 450) are located in the optineurin promoter (SEQ ID NO: 1)at the respective residues indicated in Table 2. In accordance with theembodiments of the present invention, transcription of optineurinmolecules can be effected by agents capable of altering the biochemicalproperties or concentration of NFkB 3 or its homologues, including, butnot limited to, the concentration of NFkB or its homologues bound to anupstream motif or cis element. Such agents can be used in the study ofglaucoma pathogenesis. In another embodiment, such agents can also beused in the study of glaucoma prognosis. In another embodiment suchagents can be used in the treatment of glaucoma.

[0067] An NF1 motif or cis element has been identified which recognizesa family of at least six proteins. Courtois et al., Nucleic Acid Res.18: 57-64 (1990); Mul et al., J. Virol. 64: 5510-5518 (1990); Rossi etal., Cell 52: 405-414 (1988); Gounari et al., EMBO J. 10: 559-566(1990); Goyal et al., Mol. Cell Biol. 10: 1041-1048 (1990); Mermond etal., Nature 332: 557-561 (1988); Gronostajski et al., Molecular andCellular Biology 5: 964-971 (1985); Hennighausen et al., EMBO J. 5:1367-1371 (1986); Chodosh et al., Cell 53: 11-24 (1988). The NF1 proteinwill bind to an NF1 motif or cis element either as a dimer (if the motifis palindromic) or as an single molecule (if the motif is notpalindromic). The NF1 protein is induced by TGFβ. Faisst and Meyer,Nucleic Acid Research 20: 3-26 (1992). Sequences corresponding to anupstream motif or cis element capable of binding NF1 (SEQ ID NOs: 117and 194) are located in the optineurin promoter (SEQ ID NO: 1) at therespective residues indicated in Table 2. In accordance with theembodiments of the present invention, transcription of optineurinmolecules can be effected by agents capable of altering the biochemicalproperties or concentration of NF1 or its homologues, including, but notlimited to, the concentration of NF1 or its homologues bound to anupstream motif or cis element. Such agents can be used in the study ofglaucoma pathogenesis. In another embodiment, such agents can also beused in the study of glaucoma prognosis. In another embodiment suchagents can be used in the treatment of glaucoma.

[0068] Sequences corresponding to an upstream motif or cis elementcapable of binding zinc (SEQ ID NOs: 217, 223 and 417) are located inthe optineurin promoter (SEQ ID NO: 1) at the respective residuesindicated in Table 2. In accordance with the embodiments of the presentinvention, transcription of optineurin molecules can be effected byagents capable of altering the biochemical properties or concentrationof zinc. Such agents can be used in the study of glaucoma pathogenesis.In another embodiment, such agents can also be used in the study ofglaucoma prognosis. In another embodiment such agents can be used in thetreatment of glaucoma.

[0069] Human transcription factor activator protein 2 (AP2) is atranscription factor that has been shown to bind to Sp1, nuclear factor1 (NF1) and simian virus 40 transplantation (SV40 T) antigen bindingsites. It is developmentally regulated. Williams and Tijan, Gene Dev. 5:670-682 (1991); Mitchell et al., Genes Dev. 5: 105-119 (1991); Coutoiset al., Nucleic Acid Research 18: 57-64 (1990); Comb et al., NucleicAcid Research 18: 3975-3982 (1990); Winings et al., Nucleic AcidResearch 19: 3709-3714 (1991). Sequences corresponding to an upstreammotif or cis element capable of binding AP2 (SEQ ID NOs: 327, 374, and463) are located in the optineurin promoter (SEQ ID NO: 1) at therespective residues indicated in Table 2. In accordance with theembodiments of the present invention, transcription of optineurinmolecules can be effected by agents capable of altering the biochemicalproperties or concentration of AP2 or its homologues, including, but notlimited to, the concentration of AP2 or its homologues bound to anupstream motif or cis element. Such agents may be useful in the study ofglaucoma pathogenesis. In another embodiment, such agents can also beused in the study of glaucoma prognosis. In another embodiment suchagents can be used in the treatment of glaucoma.

[0070] The sex-determining region of the Y chromosome gene, sry, isexpressed in the fetal mouse for a brief period, just prior to testisdifferentiation. SRY is a DNA binding protein known to bind to aCACA-rich region in the sry gene. Vriz et al., Biochemistry andMolecular Biology International 37: 1137-1146(1995). Sequencescorresponding to an upstream motif or cis element capable of binding SRY(SEQ ID NOs: 335 and 393) are located in the optineurin promoter (SEQ IDNO: 1) at the respective residues indicated in Table 2. In accordancewith the embodiments of the present invention, transcription ofoptineurin molecules can be effected by agents capable of altering thebiochemical properties or concentration of SRY or its homologues,including, but not limited to, the concentration of SRY or itshomologues bound to an upstream motif or cis element. Such agents may beuseful in the study of glaucoma pathogenesis. In another embodiment,such agents can also be used in the study of glaucoma prognosis. Inanother embodiment such agents can be used in the treatment of glaucoma.

[0071] Normal liver and differentiated hepatoma cell lines contain ahepatocyte-specific nuclear factor (HNF-1) which binds cis-actingelement sequences within the promoters of the alpha and beta chains offibrinogen and alpha 1-antitrypsin. Baumhueter et al., EMBO J. 8:2485-2493. Sequences corresponding to an HNF-1 upstream motif or ciselement (SEQ ID NOs: 126, 263, 423 and 426) are located in theoptineurin promoter (SEQ ID NO: 1) at the respective residues indicatedin Table 2. In accordance with the embodiments of the present invention,transcription of optineurin molecules can be effected by agents capableof altering the biochemical properties or concentration of HNF-1 or itshomologues, including, but not limited to, the concentration of HNF-1 orits homologues bound to an HNF-1 upstream motif or cis element. Suchagents can be used in the study of glaucoma pathogenesis. In anotherembodiment, such agents can also be used in the study of glaucomaprognosis. In another embodiment such agents can be used in thetreatment of glaucoma.

[0072] Alu repetitive elements are unique to primates and areinterspersed within the human genome with an average spacing of 4Kb.While some Alu sequences are actively transcribed by polymerase III,certain mRNA transcripts may also contain Alu-derived sequences in 5′ or3′ untranslated regions. Jurka and Mikahanljaia, J. Mol. Evolution 32:105-121 (1991); Claveria and Makalowski, Nature 371: 751-752 (1994).Sequences corresponding to an Alu upstream motif or cis element (SEQ IDNOs: 462 and 463) are located in the optineurin promoter (SEQ ID NO: 1)at residues 1002 through 1328 and 2288 through 2588, respectively, asdepicted in FIG. 3 by a dotted line above the nucleotides.

[0073] In accordance with the embodiments of the present invention,transcription of optineurin molecules can be effected by agents capableof altering the biochemical properties or concentration of nuclearfactors or their homologues, including, but not limited to, theconcentration of nuclear factors or their homologues bound to an Aluupstream motif or cis element. Such agents can be used in the study ofglaucoma pathogenesis. In another embodiment, such agents can also beused in the study of glaucoma prognosis. In another embodiment suchagents can be used in the treatment of glaucoma.

[0074] Sequences corresponding to repeat elements (SEQ ID NOs: 460 and461) are located in the optineurin promoter (SEQ ID NO: 1) at residues598 through 878, and 938 through 957, respectively, as depicted in FIG.3 by a dotted line above the nucleotides. In accordance with theembodiments of the present invention, transcription of optineurinmolecules can be effected by agents capable of altering the biochemicalproperties or concentration of nuclear factors or their homologues,including, but not limited to, the concentration of nuclear factors ortheir homologues bound to a repeat element. Such agents can be used inthe study of glaucoma pathogenesis. In another embodiment, such agentscan also be used in the study of glaucoma prognosis. In anotherembodiment such agents can be used in the treatment of glaucoma.

[0075] Agents of the invention include nucleic acid molecules. In oneaspect of the present invention the nucleic acid molecule is anoptineurin promoter. An example of an optineurin promoter is the nucleicacid sequence set forth in SEQ ID NO: 1. In a preferred aspect of thepresent invention, the optineurin promoter comprises a fragment of SEQID NO: 1 that itself comprises at least one ATG initiation codon andincludes preferably between 100 and 500 consecutive nucleotides, morepreferably between 200 and 1000 consecutive nucleotides, and mostpreferably between 500 and 5,000 consecutive nucleotides of SEQ IDNO: 1. In a particularly preferred embodiment, the optineurin promoterfragment comprises at least 150 bases upstream of the TATA-box. Morepreferably, the optineurin promoter fragment is at least 15 consecutivenucleotides but not more than 1500 consecutive nucleotides of SEQ ID NO:1 in length. In a preferred embodiment, the optineurin promoter fragmentis at least 20 consecutive nucleotides but not more than 1500consecutive nucleotides of SEQ ID NO: 1 in length.

[0076] In one embodiment the nucleic acid molecule is a DNA molecule. Inanother embodiment the nucleic acid molecule is an RNA molecule, morepreferably an mRNA molecule. In a further embodiment the nucleic acidmolecule is a double stranded molecule. In another further embodimentthe nucleic acid molecule is a single stranded molecule.

[0077] In one embodiment, the nucleic acid molecule comprises one ormore of the cis elements listed in Table 2. In another embodiment, thenucleic acid molecule comprises two or more of the cis elements listedin Table 2. In a further embodiment, the nucleic acid molecule comprisesthree, four, five, about ten, about fifteen or more, or between 3 and 3,4 and 6, 5 and 7, 6 and 9, 10 and 15 or 20 and 30 of the cis elementslisted in Table 2.

[0078] The present invention provides nucleic acid molecules thathybridize to the above-described nucleic acid molecules. Nucleic acidhybridization is a technique well known to those of skill in the art ofDNA manipulation. The hybridization properties of a given pair ofnucleic acids is an indication of their similarity or identity.

[0079] The nucleic acid molecules preferably hybridize, under low,moderate, or high stringency conditions, with a nucleic acid sequenceselected from: (1) any of SEQ ID NOs: 3 through 463. In another aspect,the nucleic acid molecules preferably hybridize, under low, moderate, orhigh stringency conditions, with a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 1 and its complement.

[0080] The hybridization conditions typically involve nucleic acidhybridization in about 0.1X to about 10X SSC (diluted from a 20X SSCstock solution containing 3 M sodium chloride and 0.3 M sodium citrate,pH 7.0 in distilled water), about 2.5X to about 5X Denhardt's solution(diluted from a 50X stock solution containing 1% (w/v) bovine serumalbumin, 1% (w/v) ficoll, and 1% (w/v) polyvinylpyrrolidone in distilledwater), about 10 mg/mL to about 100 mg/mL fish sperm DNA, and about0.02% (w/v) to about 0.1% (w/v) SDS, with an incubation at about 20° C.to about 70° C. for several hours to overnight. The stringencyconditions are preferably provided by 6X SSC, 5X Denhardt's solution,100 mg/mL fish sperm DNA, and 0.1% (w/v) SDS, with an incubation at 55°C. for several hours.

[0081] The hybridization is generally followed by several wash steps.The wash compositions generally comprise 0.1X to about 10X SSC, and0.01% (w/v) to about 0.5% (w/v) SDS with a 15 minute incubation at about20° C. to about 70° C. Preferably, the nucleic acid segments remainhybridized after washing at least one time in 0.1X SSC at 65° C. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0 X SSC at 50° C. to a high stringency ofabout 0.2 X SSC at 65° C. In addition, the temperature in the wash stepcan be increased from low stringency conditions at room temperature,about 22° C., to high stringency conditions at about 65° C. Bothtemperature and salt may be varied, or either the temperature or thesalt concentration may be held constant while the other variable ischanged.

[0082] Low stringency conditions may be used to select nucleic acidsequences with lower sequence identities to a target nucleic acidsequence. One may wish to employ conditions such as about 6.0 X SSC toabout 10 X SSC, at temperatures ranging from about 20° C. to about 55°C., and preferably a nucleic acid molecule will hybridize to one or moreof the above-described nucleic acid molecules under low stringencyconditions of about 6.0 X SSC and about 45° C. In a preferredembodiment, a nucleic acid molecule will hybridize to one or more of theabove-described nucleic acid molecules under moderately stringentconditions, for example at about 2.0 X SSC and about 65° C. In aparticularly preferred embodiment, a nucleic acid molecule of thepresent invention will hybridize to one or more of the above-describednucleic acid molecules under high stringency conditions such as 0.2 XSSC and about 65° C.

[0083] In an alternative embodiment, the nucleic acid molecule comprisesa nucleic acid sequence that is greater than 85% identical, and morepreferably greater than 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99% identical to a nucleic acid sequence of the presentinvention, preferably one selected from the group consisting of SEQ IDNO: 1, fragments of SEQ ID NO: 1 that comprise at least 20 consecutivenucleotides but not more than 1500 consecutive nucleotides of thesequence of SEQ ID NO: 1, and complements thereof.

[0084] The percent identity is preferably determined using the “BestFit” or “Gap” program of the Sequence Analysis Software Package™(Version 10; Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis.). “Gap” utilizes the algorithm ofNeedleman and Wunsch to find the alignment of two sequences thatmaximizes the number of matches and minimizes the number of gaps.“BestFit” performs an optimal alignment of the best segment ofsimilarity between two sequences and inserts gaps to maximize the numberof matches using the local homology algorithm of Smith and Waterman. Thepercent identity calculations may also be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (defaultparameters, DNASTAR Inc., Madison, Wis.). The percent identity is mostpreferably determined using the “Best Fit” program using defaultparameters.

[0085] The present invention also provides nucleic acid moleculefragments that hybridize to the above-described nucleic acid moleculesand complements thereof, fragments of nucleic acid molecules thatexhibit greater than 80%, 85%, 90%, 95% or 99% sequence identity with anucleic acid molecule of the present invention.

[0086] Fragment nucleic acid molecules may consist of significantportion(s) of, or indeed most of, the nucleic acid molecules of theinvention. In an embodiment, the fragments are between 3000 and 1000consecutive nucleotides, 1800 and 150 consecutive nucleotides, 1500 and500 consecutive nucleotides, 1300 and 250 consecutive nucleotides, 1000and 200 consecutive nucleotides, 800 and 150 consecutive nucleotides,500 and 100 consecutive nucleotides, 300 and 75 consecutive nucleotides,100 and 50 consecutive nucleotides, 50 and 25 consecutive nucleotides,or 20 and 10 consecutive nucleotides long of a nucleic molecule of thepresent invention.

[0087] In another embodiment, the fragment comprises at least 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 750 consecutivenucleotides of a nucleic acid sequence of the present invention. Inanother embodiment, the fragment comprises at least 12, 15, 18, 20, 25,50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450 but not more 500,550, 600, 650, 700, 750, 800, 1000, 1200, 1400, or 1500 consecutivenucleotides of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1 and complements thereof.

[0088] Any of a variety of methods may be used to obtain one or more ofthe above-described nucleic acid molecules. Automated nucleic acidsynthesizers may be employed for this purpose. In lieu of suchsynthesis, the disclosed nucleic acid molecules may be used to define apair of primers that can be used with the polymerase chain reaction toamplify and obtain any desired nucleic acid molecule or fragment.

[0089] Short nucleic acid sequences having the ability to specificallyhybridize to complementary nucleic acid sequences may be produced andutilized in the present invention, e.g., as probes to identify thepresence of a complementary nucleic acid sequence in a given sample.Alternatively, the short nucleic acid sequences may be used asoligonucleotide primers to amplify or mutate a complementary nucleicacid sequence using PCR technology. These primers may also facilitatethe amplification of related complementary nucleic acid sequences (e.g.,related sequences from other species).

[0090] Use of these probes or primers may greatly facilitate theidentification of transgenic cells or organisms which contain thepresently disclosed promoters and structural nucleic acid sequences.Such probes or primers may also, for example, be used to screen cDNA orgenomic libraries for additional nucleic acid sequences related to orsharing homology with the presently disclosed promoters and structuralnucleic acid sequences. The probes may also be PCR probes, which arenucleic acid molecules capable of initiating a polymerase activity whilein a double-stranded structure with another nucleic acid.

[0091] A primer or probe is generally complementary to a portion of anucleic acid sequence that is to be identified, amplified, or mutatedand of sufficient length to form a stable and sequence-specific duplexmolecule with its complement. The primer or probe preferably is about 10to about 200 nucleotides long, more preferably is about 10 to about 100nucleotides long, even more preferably is about 10 to about 50nucleotides long, and most preferably is about 14 to about 30nucleotides long.

[0092] The primer or probe may, for example without limitation, beprepared by direct chemical synthesis, by PCR (U.S. Pat. Nos. 4,683,195and 4,683,202), or by excising the nucleic acid specific fragment from alarger nucleic acid molecule. Various methods for determining thestructure of PCR probes and PCR techniques exist in the art.Computer-generated searches using programs such as Primer3(www-genome.wi.mit. edu/cgi-bin/primer/primer3.cgi), STSPipeline(www-genome.wi.mit.edu/cgi-bin/www-STS_Pipeline), or GeneUp (Pesole etal., BioTechniques 25:112-123, 1998), for example, can be used toidentify potential PCR primers.

[0093] Nucleic acid agents of the present invention may also be employedto obtain other optineurin nucleic acid molecules. Such moleculesinclude the optineurin-encoding nucleic acid molecules of non-humananimals (particularly cats, monkeys, rodents and dogs), fragmentsthereof, and promoters and flanking sequences. Such molecules canreadily be obtained by using the above-described primers to screen cDNAor genomic libraries obtained from non-human species. Methods forforming such libraries are known in the art.

[0094] Any of the nucleic acid agents of the invention may be linkedwith additional nucleic acid sequences to encode fusion proteins. Theadditional nucleic acid sequence preferably encodes at least one aminoacid, peptide, or protein. Many possible fusion combinations exist. Forinstance, the fusion protein may provide a “tagged” epitope tofacilitate detection of the fusion protein, such as GST, GFP, FLAG, orpolyHIS. Such fusions preferably encode between 1 and 50 amino acids,more preferably between 5 and 30 additional amino acids, and even morepreferably between 5 and 20 amino acids.

[0095] Alternatively, the fusion may provide regulatory, enzymatic, cellsignaling, or intercellular transport functions. For example, a sequenceencoding a signal peptide may be added to direct a fusion protein to aparticular organelle within a eukaryotic cell. Such fusion partnerspreferably encode between 1 and 1000 additional amino acids, morepreferably between 5 and 500 additional amino acids, and even morepreferably between 10 and 250 amino acids.

[0096] The above-described protein or peptide molecules may be producedvia chemical synthesis, or more preferably, by expression in a suitablebacterial or eukaryotic host. Suitable methods for expression aredescribed by Sambrook et al., supra, or similar texts. Fusion protein orpeptide molecules of the invention are preferably produced viarecombinant means. These proteins and peptide molecules may bederivatized to contain carbohydrate or other moieties (such as keyholelimpet hemocyanin, etc.).

[0097] B. Recombinant Vectors and Constructs

[0098] Exogenous genetic material may be transferred into a host cell byuse of a vector or construct designed for such a purpose. Preferredexogenous genetic material is a nucleic acid molecule of the presentinvention, more preferred exogenous genetic material is an optineurinpromoter sequence, and even more preferred exogenous genetic material isa nucleic acid molecule comprising SEQ ID NO: 1.

[0099] Any of the nucleic acid sequences described above may be providedin a recombinant vector. As used herein, “vector” refers to a plasmid,cosmid, bacteriophage, BAC, YAC, or virus that carries exogenous DNAinto a host organism. A plasmid may be a linear or a closed circularplasmid. The vector system may be a single vector or plasmid or two ormore vectors or plasmids which together contain the total DNA to beintroduced into the genome of the host. Means for preparing recombinantvectors are well known in the art.

[0100] Vectors suitable for replication in mammalian cells may includeviral replicons, or sequences which insure integration of theappropriate sequences encoding HCV epitopes into the host genome. Forexample, another vector used to express foreign DNA is vaccinia virus.Such heterologous DNA is generally inserted into a gene which isnon-essential to the virus, for example, the thymidine kinase gene (tk),which also provides a selectable marker. Expression of the HCVpolypeptide then occurs in cells or animals which are infected with thelive recombinant vaccinia virus.

[0101] In general, plasmid vectors containing replicon and controlsequences that are derived from species compatible with the host cellare used in connection with bacterial hosts. The vector ordinarilycarries a replication site, as well as marking sequences that arecapable of providing phenotypic selection in transformed cells. Forexample, E. coli is typically transformed using pBR322, which containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid or phage, also generally contains, or is modified tocontain, promoters that can be used by the microbial organism forexpression of the selectable marker genes.

[0102] A construct or vector may include a promoter, e.g., a recombinantvector typically comprises, in a 5′ to 3′ orientation: a promoter todirect the transcription of a nucleic acid sequence of interest and anucleic acid sequence of interest. Suitable promoters include, but arenot limited to, those described herein. The recombinant vector mayfurther comprise a 3′ transcriptional terminator, a 3′ polyadenylationsignal, other untranslated nucleic acid sequences, transit and targetingnucleic acid sequences, selectable markers, enhancers, and operators, asdesired.

[0103] The vector may be an autonomously replicating vector, i.e., avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication. Forautonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Alternatively, the vector may be one which, whenintroduced into the cell, is integrated into the genome and replicatedtogether with the chromosome(s) into which it has been integrated. Thisintegration may be the result of homologous or non-homologousrecombination.

[0104] Integration of a vector or nucleic acid into the genome byhomologous recombination, regardless of the host being considered,relies on the nucleic acid sequence of the vector. Typically, the vectorcontains nucleic acid sequences for directing integration by homologousrecombination into the genome of the host. These nucleic acid sequencesenable the vector to be integrated into the host cell genome at aprecise location or locations in one or more chromosomes. To increasethe likelihood of integration at a precise location, there should bepreferably two nucleic acid sequences that individually contain asufficient number of nucleic acids, preferably 400 bp to 1500 bp, morepreferably 800 bp to 1000 bp, which are highly homologous with thecorresponding host cell target sequence. This enhances the probabilityof homologous recombination. These nucleic acid sequences may be anysequence that is homologous with a host cell target sequence and,furthermore, may or may not encode proteins.

[0105] Promoters

[0106] In addition to the optineurin promoters described herein, otherpromoter sequences can be utilized in a vector or other nucleic acidmolecule. In a preferred aspect, the promoter is operably linked toanother nucleic acid molecule. The promoters may be selected on thebasis of the cell type into which the vector will be inserted. Thepromoters may also be selected on the basis of their regulatoryfeatures, e.g., enhancement of transcriptional activity, inducibility,tissue specificity, and developmental stage-specificity. Additionalpromoters that may be utilized are described, for example, in Bernoistand Chambon, Nature 290:304-310 (1981); Yamamoto et al., Cell 22:787-797(1980); Wagner et al., PNAS 78:1441-1445 (1981); Brinster et al., Nature296:39-42 (1982).

[0107] Suitable promoters for mammalian cells are also known in the artand include viral promoters, such as those from Simian Virus 40 (SV40),Rous sarcoma virus (RSV), adenovirus (ADV), cytomegalovirus (CMV), andbovine papilloma virus (BPV), as well as mammalian cell-derivedpromoters. Other preferred promoters include the hematopoietic stemcell-specific, e.g., CD34, glucose-6-phosphotase, interleukin-1 alpha,CD11c integrin gene, GM-CSF, interleukin-5R alpha, interleukin-2, c-fos,h-ras and DMD gene promoters. Other promoters include the herpesthymidine kinase promoter, and the regulatory sequences of themetallothionein gene.

[0108] Inducible promoters suitable for use with bacteria hosts includethe β-lactamase and lactose promoter systems, the arabinose promotersystem, alkaline phosphatase, a tryptophan (trp) promoter system andhybrid promoters such as the tac promoter. However, other knownbacterial inducible promoters are suitable. Promoters for use inbacterial systems also generally contain a Shine-Dalgarno sequenceoperably linked to the DNA encoding the polypeptide of interest.

[0109] Additional Nucleic Acid Sequences of Interest

[0110] The recombinant vector may also contain one or more additionalnucleic acid sequences of interest. These additional nucleic acidsequences may generally be any sequences suitable for use in arecombinant vector. Such nucleic acid sequences include, withoutlimitation, any of the nucleic acid sequences, and modified formsthereof, described above. The additional nucleic acid sequences may alsobe operably linked to any of the above described promoters. The one ormore additional nucleic acid sequences may each be operably linked toseparate promoters. Alternatively, the additional nucleic acid sequencesmay be operably linked to a single promoter (i.e. a single operon).

[0111] The additional nucleic acid sequences include, withoutlimitation, those encoding gene products which are toxic to a cell suchas the diptheria A gene product.

[0112] Alternatively, the additional nucleic acid sequence may bedesigned to down-regulate a specific nucleic acid sequence. This istypically accomplished by operably linking the additional nucleic acidsequence, in an antisense orientation, with a promoter. One of ordinaryskill in the art is familiar with such antisense technology. Any nucleicacid sequence may be negatively regulated in this manner. Preferabletarget nucleic acid sequences include SEQ ID NOs: 3 through 463.

[0113] Selectable and Screenable Markers

[0114] A vector or construct may also include a selectable marker.Selectable markers may also be used to select for organisms or cellsthat contain the exogenous genetic material. Examples of such include,but are not limited to: a neo gene, which codes for kanamycin resistanceand can be selected for using kanamycin, GUS, green fluorescent protein(GFP), neomycin phosphotransferase II (nptII), luciferase (LUX), or anantibiotic resistance coding sequence.

[0115] A vector or construct may also include a screenable marker.Screenable markers may be used to monitor expression. Exemplaryscreenable markers include: a β-glucuronidase or uidA gene (GUS) whichencodes an enzyme for which various chromogenic substrates are known; aβ-lactamase gene, a gene which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a luciferase gene; a tyrosinase gene, which encodes anenzyme capable of oxidizing tyrosine to DOPA and dopaquinone which inturn condenses to melanin; and α-galactosidase, which will turn achromogenic α-galactose substrate.

[0116] Included within the terms “selectable or screenable marker genes”are also genes which encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers which encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes which canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins which are detectable,(e.g., by ELISA), or small active enzymes which are detectable inextracellular solution (e.g., α-amylase, β-lactamase, phosphinothricintransferase). Other possible selectable and/or screenable marker geneswill be apparent to those of skill in the art.

[0117] C. Transgenic Organisms, Transformed and Transfected Host Cells

[0118] One or more of the nucleic acid molecules or recombinant vectorsof the invention may be used in transformation or transfection. Forexample, exogenous genetic material may be transferred into a cell ororganism. In a preferred embodiment, the exogenous genetic materialincludes a nucleic acid molecule of the present invention, preferably anucleic acid molecule of an optineurin promoter. In another preferredembodiment, the nucleic acid molecule has a sequence selected from thegroup consisting of SEQ ID NO: 1, fragments of SEQ ID NO: 1 thatcomprise at least 20 consecutive nucleotides but not more than 1500consecutive nucleotides of the sequence of SEQ ID NO: 1, and complementsthereof.

[0119] The invention is also directed to transgenic or transfectedorganisms and transformed or transfected host cells which comprise, in a5′ to 3′ orientation, a promoter operably linked to a heterologousnucleic acid sequence of interest. Additional nucleic acid sequences maybe introduced into the organism or host cell, such as 3′ transcriptionalterminators, 3′ polyadenylation signals, other untranslated nucleic acidsequences, signal or targeting sequences, selectable markers, enhancers,and operators. Preferred nucleic acid sequences of the presentinvention, including recombinant vectors, structural nucleic acidsequences, promoters, and other regulatory elements, are describedherein. Another embodiment of the invention is directed to a method ofproducing such transgenic organisms which generally comprises the stepsof selecting a suitable organism, transforming the organism with arecombinant vector, and obtaining the transformed organism.

[0120] Transfer of a nucleic acid that encodes a protein can result inexpression or overexpression of that protein in a transformed cell ortransgenic organism. One or more of the proteins or fragments thereofencoded by nucleic acid molecules of the invention may be overexpressedin a transformed cell or transgenic organism. Such expression oroverexpression may be the result of transient or stable transfer of theexogenous genetic material.

[0121] The expressed protein may be detected using methods known in theart that are specific for the particular protein or fragment. Thesedetection methods may include the use of specific antibodies, formationof an enzyme product, or disappearance of an enzyme substrate. Forexample using the antibodies to the protein. The techniques of enzymeassay and immunoassay are well known to those skilled in the art.

[0122] The resulting protein may be recovered by methods known in thearts. For example, the protein may be recovered from the nutrient mediumby procedures including, but not limited to, centrifugation, filtration,extraction, spray-drying, evaporation, or precipitation. The recoveredprotein may then be further purified by a variety of chromatographicprocedures, e.g., ion exchange chromatography, gel filtrationchromatography, affinity chromatography, or the like. Reverse-phase highperformance liquid chromatography (RP-HPLC), optionally employinghydrophobic RP-HPLC media, e.g., silica gel, further purify the protein.Combinations of methods and means can also be employed to provide asubstantially purified recombinant polypeptide or protein.

[0123] Technology for introduction of nucleic acids into cells is wellknown to those of skill in the art. Common methods include chemicalmethods, microinjection, electroporation (U.S. Pat. No. 5,384,253),particle acceleration, viral vectors, and receptor-mediated mechanisms.Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts and regeneration of thecell wall. The various techniques for transforming mammalian cells arealso well known.

[0124] There are many methods for introducing transforming DNA segmentsinto cells, but not all are suitable for delivering DNA to eukaryoticcells. Suitable methods include virtually any method by which DNA can beintroduced into a cell, such as by direct delivery of DNA, bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, by chemical transfection, by lipofection or liposome-mediatedtransfection, by calcium chloride-mediated DNA uptake, etc. In certainembodiments, acceleration methods are preferred and include, forexample, microprojectile bombardment and the like.

[0125] A transformed or transfected host cell may generally be any cellwhich is compatible with the present invention. A transformed ortransfected host organism or cell can be or derived from a cell ororganism such as a mammalian cell, mammal, fish cell, fish, bird cell,bird, fungal cell, fungus, or bacterial cell. Preferred host andtransformants include: fungal cells such as Aspergillus, yeasts,mammals, particularly murine, bovine and porcine, insects, bacteria, andalgae. Methods to transform and transfect such cells or organisms areknown in the art. See, e.g., EP 238023; Becker and Guarente, in: Abelsonand Simon (eds.), Guide to Yeast Genetics and Molecular Biology, MethodsEnzymol. 194: 182-187, Academic Press, Inc., New York; Bennett andLaSure (eds.), More Gene Manipulations in Fungi, Academic Press, Calif.,1991; Hinnen et al., PNAS 75:1920, 1978; Ito et al., J. Bacteriology153:163, 1983; Malardier et al., Gene 78:147-156, 1989; Yelton et al.,PNAS 81:1470-1474, 1984. Mammalian cell lines available as hosts forexpression are known in the art and include many immortalized cell linesavailable from the American Type Culture Collection (ATCC, Manassas,Va.), such as HeLa cells, Chinese hamster ovary (CHO) cells, babyhamster kidney (BHK) cells and a number of other cell lines.Non-limiting examples of suitable mammalian host cell lines includethose shown below in Table 3. TABLE 3 Mammalian Host Cell Lines HostCell Origin Source HepG-2 Human Liver Hepatoblastoma ATCC HB 8065 CV-1African Green Monkey Kidney ATCC CCL 70 LLC-MK₂ Rhesus Monkey KidneyATCC CCL 7 3T3 Mouse Embryo Fibroblasts ATCC CCL 92 AV12-664 SyrianHamster ATCC CRL 9595 HeLa Human Cervix Epitheloid ATCC CCL 2 RPMI8226Human Myeloma ATCC CCL 155 H4IIEC3 Rat Hepatoma ATCC CCL 1600 C127IMouse Fibroblast ATCC CCL 1616 293 Human Embryonal Kidney ATCC CRL 1573HS-Sultan Human Plasma Cell Plasmocytoma ATCC CCL 1484 BHK-21 BabyHamster Kidney ATCC CCL 10 HTM Human Trabecular Meshwork Stamer*hTERT-RPE1 Human Retinal Pigment Clontech^(†) Epithelial Cells HCE HumanCorneal Epithelium LSU Eye Center^(‡) B-3 Human Eye CRL-11421 CHO-K1Chinese Hamster Ovary ATCC CCL 61

[0126] A fungal host cell may, for example, be a yeast cell, a fungi, ora filamentous fungal cell. In one embodiment, the fungal host cell is ayeast cell, and in a preferred embodiment, the yeast host cell is a cellof the species of Candida, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Pichia and Yarrowia. In another embodiment, thefungal host cell is a filamentous fungal cell, and in a preferredembodiment, the filamentous fungal host cell is a cell of the species ofAcremonium, Aspergillus, Fusarium, Humicola, Myceliophthora, Mucor,Neurospora, Penicillium, Thielavia, Tolypocladium and Trichoderma.

[0127] Suitable host bacteria include archaebacteria and eubacteria,especially eubacteria and most preferably Enterobacteriaceae. Examplesof useful bacteria include Escherichia, Enterobacter, Azotobacter,Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella,Serratia, Shigella, Rhizobia, Vitreoscilla and Paracoccus. Suitable E.coli hosts include E. coli W3110 (ATCC 27325), E. coli 294 (ATCC 31446),E. coli B and E. coli X1776 (ATCC 31537) (American Type CultureCollection, Manassas, Va.). Mutant cells of any of the above-mentionedbacteria may also be employed. These hosts may be used with bacterialexpression vectors such as E. coli cloning and expression vectorBluescript™ (Stratagene, La Jolla, Calif.); pIN vectors (U.S. Pat. No.5,426,050), and pGEX vectors (Promega, Madison, Wis.), which may be usedto express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST).

[0128] Preferred insect host cells are derived from Lepidopteran insectssuch as Spodoptera frugiperda or Trichoplusia ni. The preferredSpodoptera frugiperda cell line is the cell line Sf9 (ATCC CRL 1711).Other insect cell systems, such as the silkworm B. mori may also beused. These host cells are preferably used in combination withBaculovirus expression vectors (BEVs), which are recombinant insectviruses in which the coding sequence for a chosen foreign gene has beeninserted behind a baculovirus promoter in place of the viral gene, e.g.,polyhedrin (U.S. Pat. No. 4,745,051).

[0129] One aspect of the present invention relates to transgenicnon-human animals having germline and/or somatic cells in which thebiological activity of one or more genes are altered by a chromosomallyincorporated transgene. In a preferred embodiment, the transgene encodesan antisense transcript which, when transcribed from the transgene,hybridizes with a portion of the optineurin promoter sequence, andinhibits expression of the optineurin gene.

[0130] In one embodiment, the present invention provides a desirednon-human animal or an animal (including human) cell which contains apredefined, specific and desired alteration rendering the non-humananimal or animal cell predisposed to glaucoma. Specifically, theinvention pertains to a genetically altered non-human animal (mostpreferably, a mouse), or a cell (either non-human animal or human) inculture, that expresses an antisense sequence directed to the optineurinpromoter. Animals that express an antisense sequence directed to theoptineurin promote may exhibit a higher susceptibility to glaucoma orother ophthalmic disorders. By way of example, a genetically alteredmouse of this type is able to serve as a model for hereditary glaucomasand as a test animal for glaucoma studies. Non-human animals or animalcells that express an antisense sequence directed to the optineurinpromoter are able to serve as a glaucoma model. The inventionadditionally pertains to the use of such non-human animals or animalcells. Furthermore, it is contemplated that cells of the transgenicanimals of the present invention can include other transgenes.

[0131] D. Inhibition of Gene Expression

[0132] In one aspect the activity or expression of an optineurinmolecule is reduced by affecting the activity of the optineurinpromoter. In a preferred aspect, the activity or expression of anoptineurin molecule is reduced by greater than 50%, 60%, 70%, 80% or 90%by the introduction into a recipient cell or host of an agent of theinvention.

[0133] Antisense approaches are a way of preventing or reducing genefunction by targeting the genetic material. The objective of theantisense approach is to use a sequence complementary to the target geneor its promoter to block its expression and create a mutant cell line ororganism in which the level of a single chosen protein is selectivelyreduced or abolished. Antisense techniques have several advantages overother ‘reverse genetic’ approaches. The site of inactivation and itsdevelopmental effect can be manipulated by the choice of promoter forantisense genes or by the timing of external application ormicroinjection. Antisense can manipulate its specificity by selectingeither unique regions of the target gene or regions where it shareshomology to other related genes.

[0134] Under one embodiment, the process involves the introduction andexpression of an antisense gene sequence. Such a sequence is one inwhich part or all of the normal gene sequences are placed under apromoter in inverted orientation so that the ‘wrong’ or complementarystrand is transcribed into a noncoding antisense RNA that hybridizeswith the target mRNA and interferes with its expression. An antisensevector can be constructed by standard procedures and introduced intocells by transformation, transfection, electroporation, microinjection,infection, etc. The type of transformation and choice of vector willdetermine whether expression is transient or stable. The promoter usedfor the antisense gene may influence the level, timing, tissue,specificity, or inducibility of the antisense inhibition.

[0135] One aspect of the invention relates to the use of nucleic acids,e.g., SEQ ID NOs: 1 through 463, fragments thereof, or sequencescomplementary thereto, in antisense therapy. As used herein, antisensetherapy refers to administration or in situ generation ofoligonucleotide molecules or their derivatives which specificallyhybridize (e.g., bind) under physiological conditions with the cellularmRNA and/or genomic DNA, thereby inhibiting transcription and/ortranslation of that gene. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, antisense therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

[0136] An antisense construct of the present invention can be delivered,for example, as an expression plasmid which, when transcribed in thecell, produces RNA which is complementary to at least a unique portionof the cellular mRNA. Alternatively, the antisense construct is anoligonucleotide probe which is generated ex vivo and which, whenintroduced into the cell, causes inhibition of expression by hybridizingwith the mRNA and/or genomic sequences of a subject nucleic acid. Sucholigonucleotide probes are preferably modified oligonucleotides whichare resistant to endogenous nucleases, e.g., exonucleases and/orendonucleases, and are therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphorothioate and methylphosphonate analogs of DNA (see also U.S.Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, generalapproaches to constructing oligomers useful in antisense therapy havebeen reviewed, for example, by Van der Krol et al., BioTechniques6:958-976 (1988); and Stein et al., Cancer Res 48:2659-2668 (1988). Withrespect to antisense DNA, oligodeoxyribonucleotides derived from thetranslation initiation site, e.g., between the −10 and +10 regions ofthe nucleotide sequence of interest, are preferred.

[0137] Antisense approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to mRNA. The antisenseoligonucleotides will bind to the mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. In the case of double-stranded antisense nucleic acids, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

[0138] Oligonucleotides that are complementary to the 5′ end of themRNA, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have recently been shown to be effective atinhibiting translation of mRNAs as well. See Wagner, Nature 372:333(1994). Therefore, oligonucleotides complementary to either the 5′ or 3′untranslated, non-coding regions of a gene could be used in an antisenseapproach to inhibit translation of endogenous mRNA. Oligonucleotidescomplementary to the 5′ untranslated region of the mRNA should includethe complement of the AUG start codon. Antisense oligonucleotidescomplementary to mRNA coding regions are typically less efficientinhibitors of translation but could also be used in accordance with theinvention. Whether designed to hybridize to the 5′, 3′, or coding regionof subject mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably less than about 100 and morepreferably less than about 50, 25, 17 or 10 nucleotides in length.

[0139] Regardless of the choice of target sequence, it is preferred thatin vitro studies are first performed to inhibit gene expression. It ispreferred that these studies utilize controls that distinguish betweenantisense gene inhibition and nonspecific biological effects ofoligonucleotides. It is also preferred that these studies compare levelsof the target RNA or protein with that of an internal control RNA orprotein. Additionally, it is envisioned that results obtained using theantisense oligonucleotide are compared with those obtained using acontrol oligonucleotide. It is preferred that the controloligonucleotide is of approximately the same length as the testoligonucleotide and that the nucleotide sequence of the oligonucleotidediffers from the antisense sequence no more than is necessary to preventspecific hybridization to the target sequence.

[0140] The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., PNAS 86:6553-6556 (1989); Lemaitre et al.,PNAS 84:648-652 (1987); WO 88/09810) or the blood-brain barrier (see,e.g., WO 89/10134), hybridization-triggered cleavage agents (See, e.g.,Krol et al., BioTechniques 6:958-976 (1988)), or intercalating agents(see, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

[0141] Antisense oligonucleotides may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxytriethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0142] Antisense oligonucleotides may also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose. The antisenseoligonucleotide can also contain a neutral peptide-like backbone. Suchmolecules are termed peptide nucleic acid (PNA)-oligomers and aredescribed, e.g., in Perry-O'Keefe et al., PNAS 93:14670 (1996) and inEglom et al., Nature 365:566 (1993). One advantage of PNA oligomers istheir capability to bind to complementary DNA essentially independentlyfrom the ionic strength of the medium due to the neutral backbone of theDNA. In yet another embodiment, the antisense oligonucleotide comprisesat least one modified phosphate backbone selected from the groupconsisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0143] In yet a further embodiment, the antisense oligonucleotide is analpha-anomeric oligonucleotide. An alpha-anomeric oligonucleotide formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual beta-units, the strands run parallel to each other(Gautier et al., Nucl. Acids Res. 15:6625-6641 (1987)). Theoligonucleotide is a 2′-O-methylribonucleotide (Inoue et al., Nucl.Acids Res. 15:6131-12148 (1987)), or a chimeric RNA-DNA analogue (Inoueet al., FEBS Lett. 215:327-330 (1987)).

[0144] Antisense molecules can be delivered to cells which express thetarget nucleic acid in vivo. A number of methods have been developed fordelivering antisense DNA or RNA to cells; e.g., antisense molecules canbe injected directly into the tissue site, or modified antisensemolecules, designed to target the desired cells (e.g., antisense linkedto peptides or antibodies that specifically bind receptors or antigensexpressed on the target cell surface) can be administered systemically.

[0145] However, it is often difficult to achieve intracellularconcentrations of the antisense sufficient to suppress translation ofendogenous mRNAs. Therefore, a preferred approach utilizes a recombinantDNA construct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. The use of such aconstruct to transfect target cells in the patient will result in thetranscription of sufficient amounts of single stranded RNAs that willform complementary base pairs with the endogenous transcripts andthereby prevent translation of the target mRNA. For example, a vectorcan be introduced in viva such that it is taken up by a cell and directsthe transcription of an antisense RNA. Such a vector can remain episomalor become chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art, and can beplasmid, viral, or others known in the art for replication andexpression in mammalian cells.

[0146] Expression of the sequence encoding the antisense RNA can be byany promoter known in the art to act in mammalian, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude but are not limited to: the SV40 early promoter region, thepromoter contained in the 3′ long terminal repeat of Rous sarcoma virus,the herpes thymidine kinase promoter, the regulatory sequences of themetallothionein gene, etc. Any type of plasmid, cosmid, BAC, YAC orviral vector can be used to prepare the recombinant DNA construct whichcan be introduced directly into the tissue site; e.g., the choroidplexus or hypothalamus. Alternatively, viral vectors can be used whichselectively infect the desired tissue (e.g., for brain, herpesvirusvectors may be used), in which case administration may be accomplishedby another route (e.g., systemically).

[0147] Antisense RNA, DNA, and ribozyme molecules of the invention maybe prepared by any method known in the art for the synthesis of DNA andRNA molecules. These include techniques for chemically synthesizingoligodeoxyribonucleotides and oligoribonucleotides well known in the artsuch as for example solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding the antisense RNA molecule. SuchDNA sequences may be incorporated into a wide variety of vectors whichincorporate suitable RNA polymerase promoters such as the T7 or SP6polymerase promoters. Alternatively, antisense cDNA constructs thatsynthesize antisense RNA constitutively or inducibly, depending on thepromoter used, can be introduced stably into cell lines.

[0148] Moreover, various well-known modifications to nucleic acidmolecules may be introduced as a means of increasing intracellularstability and half-life. Possible modifications include but are notlimited to the addition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

[0149] Endogenous gene expression can be reduced by inactivating or“knocking out” the gene or its promoter using targeted homologousrecombination. (E.g. see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321(1989)). For example, a mutant, non-functional gene (or acompletely unrelated DNA sequence) flanked by DNA homologous to theendogenous gene (either the coding regions or regulatory regions of thegene) can be used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express that gene in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the gene.

[0150] E. Pharmaceutical Compositions

[0151] Pharmaceutical compositions can comprise polynucleotides of thepresent invention. The pharmaceutical compositions will comprise atherapeutically effective amount of nucleic acid molecules of thepresent invention.

[0152] The term “therapeutically effective amount” as used herein refersto an amount of a therapeutic agent to treat, ameliorate, or prevent adesired disease or condition, or to exhibit a detectable therapeutic orpreventative effect. The effect can be detected by, for example,chemical markers or antigen levels. Therapeutic effects also includereduction in physical symptoms, such as decreased body temperature. Theprecise effective amount for a subject will depend upon the subject'ssize and health, the nature and extent of the condition, and thetherapeutics or combination of therapeutics selected for administration.Thus, it is not useful to specify an exact effective amount in advance.However, the effective amount for a given situation can be determined byroutine experimentation and is within the judgment of the clinician.

[0153] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0154] A therapeutically effective dose refers to that amount of activeingredient, for example, an optineurin promoter molecule or fragmentsthereof, antibodies of an optineurin promoter molecule, agonists,antagonists or inhibitors of the optineurin promoter, which amelioratesthe symptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED50/LD50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0155] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0156] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc. For purposes of thepresent invention, an effective dose will be from about 0.01 mg/kg to 50mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in theindividual to which it is administered.

[0157] There is a wide variety of suitable formulations ofpharmaceutical compositions of the present invention (see, e. g.,Remington's Pharmaceutical Sciences, 17th ed. 1985). Formulationssuitable for administration include aqueous and non-aqueous solutions,isotonic sterile solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

[0158] A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, and other therapeutic agents. Theterm refers to any pharmaceutical carrier that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, and which may be administered without undue toxicity.Suitable carriers may be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art.

[0159] Pharmaceutically acceptable carriers in therapeutic compositionsmay contain liquids such as water, saline, glycerol and ethanol. Otherpharmaceutically acceptable carriers include, but are not limited to,gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols,gelatin, carbohydrates such as lactose, amylose or starch, dextrose,magnesium stearate, talc, silicic acid, viscous paraffin, fatty acidesters, hydroxmethylcellulose, polyvinyl pyrrolidone, as well ascombinations thereof. Additionally, auxiliary substances, such aswetting or emulsifying agents, lubricants, preservatives, stabilizers,pH buffering substances, coloring, flavoring and the like, may bepresent in such vehicles.

[0160] Typically, the therapeutic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection may also be prepared. Liposomes are included within thedefinition of a pharmaceutically acceptable carrier. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Solutions and suspensions can be preparedfrom sterile powders, granules, and tablets.

[0161] Pharmaceutically acceptable salts can be used therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like.Pharmaceutically acceptable excipients can also be used therein.

[0162] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions that can be used in themethods of treatment. Optionally associated with such container(s) canbe a notice or leaflet in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice or leaflet reflects approval by the agency ofmanufacture, use, or sale for human administration. The pack or kit cancontain a leaflet or be labeled with information regarding mode ofadministration, sequence of drug administration (e.g., separately,sequentially, or concurrently), or the like. The pack or kit may alsocontain means for reminding the patient to take the therapy. The pack orkit may be a single unit dosage, a plurality of unit dosages, or acombination therapy.

[0163] In particular, the agents can be separated, mixed together in anycombination, or present in a single vial or tablet. Agents assembled ina blister pack or other dispensing means is preferred. For the purposeof this invention, unit dosage is intended to mean a dosage that isdependent on the individual pharmacodynamics of each agent andadministered in FDA approved dosages in standard time courses.

[0164] Delivery Methods

[0165] Once formulated, the pharmaceuticals compositions of theinvention can be (1) administered directly to the subject; (2) deliveredex vivo, to cells derived from the subject; or (3) delivered in vitrofor expression of recombinant proteins.

[0166] Methods for direct delivery of the compositions include, but arenot limited to, subcutaneous, intraperitoneal, intraocular, intranasal,intravenous, intramuscular, intradermal, oral, intranasal, topical,intravesical, intrathecal, or delivered to the interstitial space of atissue. In a preferred embodiment, the composition is introducedintraocularly by, for example, eye drops. Other modes of administrationinclude oral and pulmonary administration, suppositories, andtransdermal applications, needles, and gene guns or hyposprays. Dosagetreatment may be a single dose schedule or a multiple dose schedule.

[0167] Methods for the ex vivo delivery and reimplantation oftransformed cells into a subject are known in the art and described ine.g., WO 93/14778. Examples of cells useful in ex vivo applicationsinclude, for example, stem cells, particularly hematopoetic, lymphcells, macrophages, dendritic cells, or tumor cells, and trabecularmeshwork cells, particularly human trabecular meshwork cells.

[0168] Generally, delivery of nucleic acids for both ex vivo and invitro applications can be accomplished by, for example, dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei, all well known in the art.

[0169] Preparation of antisense polypeptides is discussed above. Boththe dose of the antisense composition and the means of administrationare determined based on the specific qualities of the therapeuticcomposition, the condition, age, and weight of the patient, theprogression of the disease, and other relevant factors. Administrationof the therapeutic antisense agents of the invention includes local orsystemic administration, including injection, oral administration,particle gun or catheterized administration, and topical administration.Preferably, the therapeutic antisense composition contains an expressionconstruct comprising a promoter and a polynucleotide segment of at leastabout 12, 22, 25, 30, or 35 contiguous nucleotides of the antisensestrand of a nucleic acid. Within the expression construct, thepolynucleotide segment is located downstream from the promoter, andtranscription of the polynucleotide segment initiates at the promoter.

[0170] Receptor-mediated targeted delivery of therapeutic compositionscontaining an antisense polynucleotide, subgenomic polynucleotides, orantibodies to specific tissues is also used. Receptor-mediated DNAdelivery techniques are described in, for example, Findeis et al.,Trends in Biotechnol. (1993) 11:202-205; Chiou et al., (1994) GeneTherapeutics: Methods And Applications Of Direct Gene Transfer (J. A.Wolff, ed.); Wu & Wu, J. Biol. Chem. (1988) 263:621-24; Wu et al., J.Biol. Chem. (1994) 269:542-46; Zenke et al., PNAS (1990) 87:3655-59; Wuet al., J. Biol. Chem. (1991) 266:338-42. Preferably, receptor-mediatedtargeted delivery of therapeutic compositions containing antibodies ofthe invention is used to deliver the antibodies to specific tissue.

[0171] Therapeutic compositions containing antisense subgenomicpolynucleotides are administered in a range of about 100 ng to about 200mg of DNA for local administration in a gene therapy protocol.Concentration ranges of about 500 ng to about 50 mg, about 1 mg to about2 mg, about 5 mg to about 500 mg, and about 20 mg to about 100 mg of DNAcan also be used during a gene therapy protocol. Factors such as methodof action and efficacy of transformation and expression areconsiderations which will affect the dosage required for ultimateefficacy of the antisense subgenomic nucleic acids. Where greaterexpression is desired over a larger area of tissue, larger amounts ofantisense subgenomic nucleic acids or the same amounts readministered ina successive protocol of administrations, or several administrations todifferent adjacent or close tissue portions of, for example, a tumorsite, may be required to effect a positive therapeutic outcome. In allcases, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect.

[0172] For genes encoding polypeptides or proteins withanti-inflammatory activity, suitable use, doses, and administration aredescribed in U.S. Pat. No. 5,654,173. Therapeutic agents also includeantibodies to proteins and polypeptides encoded by the subject nucleicacids, as described in U.S. Pat. No. 5,654,173.

[0173] Gene Delivery

[0174] The therapeutic nucleic acids of the present invention may beutilized in gene delivery vehicles. The gene delivery vehicle may be ofviral or non-viral origin (see generally, Jolly, Cancer Gene Therapy1:51-64 (1994); Kimura, Human Gene Therapy 5:845-852 (1994); Connelly,Human Gene Therapy 1:185-193 (1995); and Kaplitt, Nature Genetics6:148-153 (1994)). Gene therapy vehicles for delivery of constructsincluding a coding sequence of a therapeutic of the invention can beadministered either locally or systemically. These constructs canutilize viral or non-viral vector approaches. Expression of such codingsequences can be induced using endogenous mammalian or heterologouspromoters. Expression of the coding sequence can be either constitutiveor regulated.

[0175] The present invention can employ recombinant retroviruses whichare constructed to carry or express a selected nucleic acid molecule ofinterest. Retrovirus vectors that can be employed include thosedescribed in EP 0415731; EP 0345242; WO 90/07936; WO 94/03622; WO93/25698; WO 93/25234; WO 93/11230; WO 93/10218; Vile and Hart, CancerRes. 53:3860-3864 (1993); Vile and Hart, Cancer Res. 53:962-967 (1993);Ram et al., Cancer Res. 53:83-88 (1993); Takamiya et al., J. Neurosci.Res. 33:493-503 (1992); Baba et al., J. Neurosurg. 79:729-735 (1993);U.S. Pat. Nos. 5,219,740 and 4,777,127; and GB Patent No. 2,200,651.Preferred recombinant retroviruses include those described in WO91/02805.

[0176] Packaging cell lines suitable for use with the above-describedretroviral vector constructs may be readily prepared (WO 95/30763 and WO92/05266), and used to create producer cell lines (also termed vectorcell lines) for the production of recombinant vector particles. Withinparticularly preferred embodiments of the invention, packaging celllines are made from human (such as HT1080 cells) or mink parent celllines, thereby allowing production of recombinant retroviruses that cansurvive inactivation in human serum.

[0177] The present invention also employs alphavirus-based vectors thatcan function as gene delivery vehicles. Such vectors can be constructedfrom a wide variety of alphaviruses, including, for example, Sindbisvirus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), RossRiver virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equineencephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCCVR-532). Representative examples of such vector systems include thosedescribed in U.S. Pat. Nos. 5,091,309; 5,217,879; and 5,185,440; and WO92/10578; WO 94/21792; WO 95/27069; WO 95/27044; and WO 95/07994.

[0178] Gene delivery vehicles of the present invention can also employparvovirus such as adeno-associated virus (AAV) vectors. Representativeexamples include the AAV vectors disclosed by Srivastava in WO 93/09239,Samulski et al., J. Vir. 63:3822-3828 (1989); Mendelson et al., Virol.(1988) 166:154-165; and Flotte et al., PNAS 90:10613-10617 (1993).

[0179] Representative examples of adenoviral vectors include thosedescribed by Berkner, Biotechniques 6:616-627 (1988); Rosenfeld et al.,Science 252:431-434 (1991); WO 93/19191; Kolls et al., PNAS 91:215-219(1994); Kass-Eisler et al., PNAS 90:11498-11502 (1993); Guzman et al.,Circulation 88:2838-2848 (1993); Guzman et al., Cir. Res. 73:1202-1207(1993); Zabner et al., Cell 75:207-216 (1993); Li et al., Hum. GeneTher. 4:403-409 (1993); Cailaud et al., Eur. J. Neurosci. 5:1287-1291(1993); Vincent et al., Nat. Genet. 5:130-134 (1993); Jaffe et al., Nat.Genet. 1:372-378 (1992); and Levrero et al., Gene 101:195-202 (1991).Exemplary adenoviral gene therapy vectors employable in this inventionalso include those described in WO 94/12649, WO 93/03769, WO 93/19191,WO 94/28938, WO 95/11984 and WO 95/00655. Administration of DNA linkedto killed adenovirus as described in Curiel, Hum. Gene Ther. 3:147-154(1992) may be employed.

[0180] Other gene delivery vehicles and methods may be employed,including polycationic condensed DNA linked or unlinked to killedadenovirus alone (Curiel, Hum. Gene Ther. 3:147-154 (1992)); ligandlinked DNA (Wu, J. Biol. Chem. 264:16985-16987 (1989)); eukaryotic celldelivery vehicles cells (U.S. Pat. No. 6,287,792); deposition ofphotopolymerized hydrogel materials; hand-held gene transfer particlegun (U.S. Pat. No. 5,149,655); ionizing radiation (U.S. Pat. No.5,206,152; WO 92/11033); and nucleic charge neutralization or fusionwith cell membranes. Additional approaches are described in Philip, Mol.Cell Biol. 14:2411-2418 (1994), and in Woffendin et al., PNAS91:11581-11585 (1994).

[0181] Naked DNA may also be employed. Exemplary naked DNA introductionmethods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by the beads. The method may be improved further by treatmentof the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.Liposomes that can act as gene delivery vehicles are described in U.S.Pat. No. 5,422,120, WO 95/13796, WO 94/23697, WO 91/14445, and EP0524968.

[0182] F. Diagnostic and Prognostic Assays

[0183] Agents of the present invention can be utilized in methods todetermine, for example, without limitation, the presence or absence of anucleic acid molecule in a sample, and the level of nucleic acidmolecule in a sample. Moreover, agents of the present invention can beutilized in methods for diagnosing glaucoma, methods for prognosingglaucoma, and methods for predicting a predisposition to glaucoma.

[0184] As used herein, the “Expression Response” manifested by a cell ortissue of an organism is said to be “altered” if it differs from theExpression Response of cells or tissues not exhibiting the phenotype. Todetermine whether a Expression Response is altered, the ExpressionResponse manifested by the cell or tissue of the organism exhibiting thephenotype is compared with that of a similar cell or tissue sample of anorganism not exhibiting the phenotype. As will be appreciated, it is notnecessary to re-determine the Expression Response of the cell or tissuesample of organisms not exhibiting the phenotype each time such acomparison is made; rather, the Expression Response of a particularorganism may be compared with previously obtained values of normalorganisms.

[0185] Also as used herein, a “tissue sample” is any sample thatcomprises more than one cell. In a preferred aspect, a tissue samplecomprises cells that share a common characteristic (e.g. derived fromneurons, epidermis, muscle etc.). Preferred cells and tissue samples maybe derived from bodily fluids including glaucomatous cell extract, fluidfrom the anterior chamber of the eye, blood, lymph, serum, amnioticfluid, and cerebrospinal fluid, or from skin, muscle, buccal orconjunctival mucosa, placenta, gastrointestinal tract or other organs. Atest sample may be derived from adults, juveniles, and fetuses. Testsamples from fetal cells or tissue can be obtained by appropriatemethods, such as by amniocentesis or chorionic villus sampling. In apreferred embodiment, a sample is derived from bodily fluids such asglaucomatous cell extract, fluid from the anterior chamber of the eye,blood, lymph, and serum.

[0186] A number of methods can be used to compare the expressionresponse between two or more samples of cells or tissue. These methodsinclude hybridization assays, such as northerns, RNAse protectionassays, and in situ hybridization. In a preferred method, the expressionresponse is compared by PCR-type assays.

[0187] An advantage of in situ hybridization over certain othertechniques for the detection of nucleic acids is that it allows aninvestigator to determine the precise spatial population. In situhybridization may be used to measure the steady-state level of RNAaccumulation. A number of protocols have been devised for in situhybridization, each with tissue preparation, hybridization and washingconditions.

[0188] In situ hybridization also allows for the localization ofproteins or mRNA within a tissue or cell. It is understood that one ormore of the molecules of the invention, preferably one or more of thenucleic acid molecules or fragments thereof of the invention or one ormore of the antibodies of the invention may be utilized to detect thelevel or pattern of a protein or mRNA thereof by in situ hybridization.

[0189] In one aspect of the present invention, an evaluation can beconducted to determine whether a optineurin nucleic acid molecule ispresent. One or more of the nucleic acid molecules of the presentinvention are utilized to detect the presence, type, or quantity of thenucleic acid molecule. Generally, such a method comprises: (a) obtainingcell or tissue sample of interest; and (b) selectively detecting thepresence or absence, or ascertaining the level of a nucleic acidmolecule.

[0190] As used herein, the term “presence” refers to when a molecule canbe detected using a particular detection methodology. Also as usedherein, the term “absence” refers to when a molecule cannot by detectedusing a particular detection methodology.

[0191] The present invention also includes and provides a method fordetermining a level or pattern of a protein in an animal cell or animaltissue comprising (A) assaying the concentration of the protein in afirst sample obtained from the animal cell or animal tissue; (B)assaying the concentration of the protein in a second sample obtainedfrom a reference animal cell or a reference animal tissue with a knownlevel or pattern of the protein; and (C) comparing the assayedconcentration of the protein in the first sample to the assayedconcentration of the protein in the second sample.

[0192] Any method for analyzing proteins can be used to detect ormeasure levels of a polypeptide. As an illustration, size differencescan be detected by Western blots of protein extracts from the twotissues. Other changes, such as expression levels and subcellularlocalization, can also be detected immunologically, using antibodies tothe corresponding protein. The expression pattern of any cell or tissuetypes can be compared. Such comparison can also occur in a temporalmanner. Another comparison can be made between difference developmentalstates of a tissue or cell sample.

[0193] More particularly, in one embodiment, mRNA in a cell or tissuesample can be detected by incubating mRNA molecules with cell or tissuesample extracts of an organism under conditions sufficient to permitnucleic acid hybridization. The detection of double-stranded probe-mRNAhybrid molecules is indicative of the presence of the mRNA; the amountof such hybrid formed is proportional to the amount of mRNA. Thus, suchprobes may be used to ascertain the level and extent of the mRNAproduction in an organism's cells or tissues. Such nucleic acidhybridization may be conducted under quantitative conditions (therebyproviding a numerical value of the amount of the mRNA present).Alternatively, the assay may be conducted as a qualitative assay thatindicates either that the mRNA is present, or that its level exceeds auser set, predefined value.

[0194] Alternatively, mRNA may be selectively detected using standardPCR or RT-PCR techniques such as those described herein. In anotherembodiment, polypeptide molecules of the present invention may beselectively detected using an immunological binding assay, e.g., an insitu binding assay. In this regard, an antibody which selectively bindsto an polypeptide of the present invention may be used. Optionally, theantibody may be labeled as described below to aid in detection.

[0195] More particularly, polypeptide molecules can be detected and/orquantified using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology: Antibodies in Cell Biology, volume 37(Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds.,7th ed. 1991). Immunological binding assays (or immunoassays) typicallyuse an antibody that specifically binds to a protein or antigen ofchoice. The antibody may be produced by any of a number of means wellknown to those of skill in the art and as described above.

[0196] Immunoassays also often use a labeling agent to specifically bindto, and label the complex formed by the antibody and antigen. Thelabeling agent may itself be one of the moieties comprising theantibody/antigen complex. Thus, the labeling agent may be a labeledpolypeptide or a labeled antibody. Alternatively, the labeling agent maybe a third moiety, such a secondary antibody, that specifically binds tothe antibody/polypeptide complex (a secondary antibody is typicallyspecific to antibodies of the species from which the first antibody isderived). Other proteins capable of specifically binding immunoglobulinconstant regions, such as protein A or protein G may also be used as thelabel agent. These proteins exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,e.g., Kronval et al., J. Immunol., 111:1401-1406 (1973); Akerstrom etal., J. Immunol., 135:2589-2542 (1985)). The labeling agent can bemodified with a detectable moiety, such as biotin, to which anothermolecule can specifically bind, such as streptavidin. A variety ofdetectable moieties are well known to those skilled in the art. Apreferred label is a fluorescent label.

[0197] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, optionally from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, antigen, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0198] Generally, immunoassays for detecting a polypeptide in a samplemay be either competitive or noncompetitive. Noncompetitive immunoassaysare assays in which the amount of antigen is directly measured. In onepreferred “sandwich” assay, for example, the antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture the polypeptide present in the testsample. The polypeptide is thus immobilized, and is then bound by alabeling agent, such as a second antibody bearing a label.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second or thirdantibody is typically modified with a detectable moiety, such as biotin,to which another molecule specifically binds, e.g., streptavidin, toprovide a detectable moiety.

[0199] Western blot (immunoblot) analysis may also used to detect andquantify the presence of polypeptide in the sample. Other assay formatsinclude liposome immunoassays (LIA), which use liposomes designed tobind specific molecules (e.g., antibodies) and release encapsulatedreagents or markers. The released chemicals are then detected accordingto standard techniques (see Monroe et al., Amer. Clin. Prod. Rev.,5:34-41 (1986)).

[0200] One of skill in the art will appreciate that it is oftendesirable to minimize non-specific binding in immunoassays.Particularly, where the assay involves an antigen or antibodyimmobilized on a solid substrate it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this technique involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine serum albumin (BSA), nonfat powdered milk, and gelatin are widelyused with powdered milk being most preferred.

[0201] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

[0202] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0203] Thus, in one aspect of the present invention, provided aremethods for diagnosing glaucoma in a sample obtained from a cell or abodily fluid by detecting a polymorphism in a promoter region of theoptineurin gene, comprising the steps of: (A) incubating underconditions permitting nucleic acid hybridization, a marker nucleic acidmolecule, the marker nucleic acid molecule having a nucleic acidsequence that specifically hybridizes to a sequence selected from thegroup consisting of SEQ ID NO: 1 and a complement thereof, and acomplementary nucleic acid molecule obtained from a sample, whereinnucleic acid hybridization between the marker nucleic acid molecule andthe complementary nucleic acid molecule permits the detection of saidpolymorphism; (B) permitting hybridization between the marker nucleicacid molecule and the complementary nucleic acid molecule; and (C)detecting the presence of the polymorphism, wherein the detection of thepolymorphism is diagnostic of glaucoma.

[0204] Also provided by the present invention are methods for prognosingglaucoma in a sample obtained from a cell or a bodily fluid by detectinga polymorphism in a promoter region of the optineurin gene, comprisingthe steps of: (A) incubating under conditions permitting nucleic acidhybridization, a marker nucleic acid molecule, the marker nucleic acidmolecule having a nucleic acid sequence that specifically hybridizes toa sequence selected from the group consisting of SEQ ID NO: 1 andcomplement thereof, and a complementary nucleic acid molecule obtainedfrom a sample, where nucleic acid hybridization between the markernucleic acid molecule and the complementary nucleic acid moleculepermits the detection of the polymorphism; (B) permitting hybridizationbetween the marker nucleic acid molecule and the complementary nucleicacid molecule; and (C) detecting the presence of the polymorphism, wherethe detection of the polymorphism is prognostic of glaucoma.

[0205] Further provided by the present invention are methods fordiagnosing or prognosing glaucoma in a sample obtained from a cell or abodily fluid by detecting a polymorphism in a promoter region of theoptineurin gene, comprising the steps of: (A) incubating underconditions permitting nucleic acid hybridization, a marker nucleic acidmolecule, the marker nucleic acid molecule having a nucleic acidsequence that specifically hybridizes to a optineurin promoter sequenceor its complement, and a complementary nucleic acid molecule obtainedfrom a sample, where nucleic acid hybridization between the markernucleic acid molecule and the complementary nucleic acid moleculepermits the detection of the polymorphism; (B) permitting hybridizationbetween the marker nucleic acid molecule and the complementary nucleicacid molecule; and (C) detecting the presence of the polymorphism, wherethe detection of the polymorphism is diagnostic or prognostic ofglaucoma.

[0206] The methods of the present invention may be used to detect asingle nucleotide polymorphism, and may further comprise a second markernucleic acid molecule.

[0207] The present invention further provides methods for detecting thepresence or absence of a SNP sequence variation in a sample containingDNA, comprising contacting a labeled nucleic acid capable of detecting asingle nucleotide polymorphism selected from table 1 with the DNA of thesample under hybridization conditions and determining the presence ofhybrid nucleic acid molecules comprising the labeled nucleic acid.

[0208] The cell or bodily fluid may comprise human trabecular meshworkcells, or may be selected from the group consisting of glaucomatous cellextract, fluid from the anterior chamber of the eye, blood, lymph, andserum. The methods may further comprise amplifying the complementarynucleic acid molecule obtained from a sample using a nucleic acidamplification method, where the nucleic acid amplification method isselected from the group consisting of polymerase chain amplification,ligase chain reaction, oligonucleotide ligation assay, thermalamplification, and transcription base amplification.

[0209] The diagnostic and prognostic methods described herein can, forexample without limitation, utilize one or more of the detection methodsdescribed herein, including but not limited to northern blot analysis,standard PCR, reverse transcription-polymerase chain reaction (RT-PCR),in situ hybridization, immunoprecipitatioon, Western blot hybridization,or immunohistochemistry.

[0210] In one aspect, the method comprises in situ hybridization with anucleic acid molecule of the present invention as a probe. This methodcomprises contacting the labeled hybridization probe with a sample of agiven type of tissue potentially containing glaucomatous orpre-glaucomatous cells as well as normal cells, and determining whetherthe probe labels some cells of the given tissue type to a degreesignificantly different (e.g., by at least a factor of two, or at leasta factor of five, or at least a factor of twenty, or at least a factorof fifty) than the degree to which it labels other cells of the sametissue type.

[0211] Alternatively, the above diagnostic assays may be carried outusing antibodies which selectively detect a polypeptide of the presentinvention. Accordingly, in one embodiment, the assay includes contactingthe proteins of the test cell with an antibody specific for apolypeptide of the present invention and determining the approximateamount of immunocomplex formation. Such a complex can be detected by anassay for example without limitation an immunohistochemical assay,dot-blot assay, and an ELISA assay.

[0212] Immunoassays are commonly used to quantitate the levels ofproteins in cell samples, and many other immunoassay techniques areknown in the art. The invention is not limited to a particular assayprocedure, and therefore is intended to include both homogeneous andheterogeneous procedures. Exemplary immunoassays which can be conductedaccording to the invention include fluorescence polarization immunoassay(FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA),nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbentassay (ELISA), and radioimmunoassay (RIA). An indicator moiety, or labelgroup, can be attached to the subject antibodies and is selected so asto meet the needs of various uses of the method which are often dictatedby the availability of assay equipment and compatible immunoassayprocedures. General techniques to be used in performing the variousimmunoassays noted above are known to those of ordinary skill in theart.

[0213] G. Modulator Screening Assays

[0214] Another aspect of the invention is directed to the identificationof agents capable of modulating one or more optineurin molecules. Suchagents are herein referred to as “modulators” or “modulating compounds”.In this regard, the invention provides assays for determining compoundsthat modulate the function and/or expression of one or more optineurinmolecules.

[0215] “Inhibitors,” “activators,” and “modulators” of optineurinmolecules are used interchangeably to refer to inhibitory, activating,or modulating molecules which can be identified using in vitro and invivo assays for optineurin activity and/or expression, e.g., ligands,agonists, antagonists, and their homologs and mimetics.

[0216] Suitable modulators include, but are not limited to, hydroxamicacids, diclofenac, MMP inhibitors, macrocyclic anti-succinatehydroxamate derivatives, anti-angiogenics, tetracyclines, steroidinactivators of metalloproteinase translation, DNA binding (minorgroove) compounds, peptide-like agents such as TIMPs, N-carboxyalkylpeptides, polyamines and glycosaminoglycans, non-steroidalanti-inflammatory drugs (NSAIDs), corticosteroids, immunosuppressiveagents, antibiotics, receptor antagonists, RNA aptamers, and antibodies.

[0217] Anti-angiogenics comprise a class of compounds including growthfactors, cytokines and peptides, which share characteristics such as theability to inhibit angiogenesis, endothelial cell proliferation,migration, tube formation and neovascularization. Preferredanti-angiogenics include endostatin and active collagen fragmentderivatives, such as arresten (a 26 kDa NC1 domain of the alpha 1 chainof type IV collagen), thrombospondin, interleukin-12, angiostatin andactive fragments and derivatives of plasminogen. See Colorado et al.,Cancer Research 60(9):2520-26 (2000); Sunamura et al., Pancreas20(3):227-33 (2000); Griscelli et al., Proceedings of the NationalAcademy of Sciences U.S.A., 95(11):6367-72 (1998). Other preferredanti-angiogenics are growth factors such as basic fibroblast growthfactor (bFGF), which may be used alone or in combination with otheranti-angiogenics such as all-trans retinoic acid to stimulate native MMPinhibitors such as tissue inhibitor of metalloproteinases-1 (TIMP-1)protein. See Bigg et al., European Journal of Biochemistry267(13):4150-56 (2000).

[0218] Hydroxamic acid-based modulators are described in U.S. Pat. No.5,240,958, and preferably have the general formula:

[0219] where R¹ represents thienyl; R² represents a hydrogen atom or aC₁-C₆ alkyl, C₁-C₆ alkenyl, phenyl(C₁-C₆) alkyl, cycloalkyl(C₁-C₆)alkylor cycloalkenyl(C₁-C₆)alkyl group; R³ represents an amino acid sidechain or a C₁-C₆ alkyl, benzyl, (C₁-C₆alkoxyl)benzyl or benzyloxy(C₁-C₆alkyl) or benzyloxy benzyl group; R⁴ represents a hydrogen atom or aC₁-C₆ alkyl group; R⁵ represents a hydrogen atom or a methyl group; n isan integer having the value 0, 1 or 2; and A represents a C₁-C₆hydrocarbon chain, optionally substituted with one or more C₁-C₆ alkyl,phenyl or substituted phenyl groups; or a salt thereof.

[0220] Other hydroxamic acid-based modulators includephosphinamide-based hydroxamic acids, peptidyl hydroxamic acidsincluding p-NH₂-Bz-Gly-Pro-D-Leu-D-Ala-NHOH (FN-439), hydroxamic acidswith a quaternary-hydroxy group, and succinate-derived hydroxamic acidsrelated to batimastat. See, e.g., Pikul et al., Journal of MedicalChemistry 42(l):87-94 (1999); Odake et al., Biochem Biophys Res Commun199(3):1442-46 (1994); Jacobson et al., Bioorganic Medical ChemistryLetters 8(7):837-42 (1998); Steimnan et al., Bioorganic MedicalChemistry Letters 8(16):2087-92 (1998). Macrocyclic anti-succinatehydroxamate derivatives can also be effective modulators. See Cherney etal., Bioorganic Medical Chemistry Letters 9(9):1279-84 (1999).Batimastat, also known as BB-94, is a relatively insoluble chemicalhaving the chemical name[2-R-[1(S*),2R*,3S*]]-N⁴-hydroxy-N¹-[2-(methylamino)-2-oxo-1-(phenylmethyl)ethyl]-2-(2-methylpropyl)-3-[(2-thienylthio)methyl]butanediamide or(2S,-3R)-5-methyl-3-[[(αS)-α-(methylcarbamoyl)phenethyl]carbamoyl]-2-[(2-thienylthio)methyl]hexanohydroxamicacid, and the formula:

[0221] Other preferred modulators include the tetracyclines, especiallyminocycline, doxycycline, and COL-3, and steroid inactivators ofmetalloproteinase translation, such as dexamethasone. See Fife et al.,Cancer Letters 153(1-2):75-8 (2000); Gilbertson-Beadling et al., CancerChemother. Pharmacol. 36(5):418-24 (1995); Greenwald et al., Journal ofRheumatology 19(6):927-38 (1992); Shapiro et al., Journal of Immunology146(8):2724-29 (1991). A further group of modulators includes DNAbinding (minor groove) compounds such as distamycin A and its sulphonicderivatives PNU145156E and PNU153429, anthramycin,pyrrolo[2,1-c][1,4]benzodiazepine (PBD) and its methyl esters, and otherpolypyrrole minor groove binders. See, e.g., Baraldi et al., Journal ofMedical Chemistry 42(25):5131-41 (1999); Possati et al., Clin. Exp.Metastasis 17(7):575-82 (1999).

[0222] The peptide-like modulators comprise a varied class of compoundsthat includes peptides, peptide mimetics, pseudopeptides, polyamines,and glycosaminoglycans. Tissue inhibitors of metalloproteinases (TIMPs)are peptides and polypeptides that inhibit the action ofmetalloproteinases and that share structural characteristics such asintrachain disulfide bonds. Preferred TIMPs include recombinant andisolated forms of natural TIMPs, including TIMP-1 (a 28.5 kDapolypeptide), TIMP-2 (a 21 kDa polypeptide), and TIMP-3 (a 24-25 kDapolypeptide), and fragments thereof that retain inhibitory function. SeeG. Murphy et al., Biochemistry 30(33):8097-102 (1991); A. N. Murphy etal., Journal of Cell Physiology 157(2):351-58 (1993); Kishnani et al.,Matrix Biology 14(6):479-88 (1995).

[0223] N-carboxyalkyl peptides are a class of peptides that includeCH₃CH₂CH₂(R,S)CH(COOH)-NH-Leu-Phe-Ala-NH₂,N-[D,L-2-isobutyl-3(N′-hydroxycarbonylamido)-propanoyl]-O-methyl-L-tyrosinemethylamide, and HSCH₂CH[CH₂CH(CH₃)₂]CO-Phe-Ala-NH₂ (SIMP). See Fini etal., Invest. Ophthalmol. Vis. Sci. 32(11):2997-3001 (1991); Stack etal., Arch. Biochem. Biophys. 287(2):240-49 (1991); Wentworth et al.,Invest. Ophthalmol. Vis. Sci. 33(7):2174-79 (1992). Other peptide-likemodulators include polyamines such as alpha-difluoromethylomithine, andglycosaminoglycans such as combretastatin and heparin. See Wallon etal., Mol. Carcinog. 11(3):138-44 (1994); Dark et al., Cancer Research 57(10):1829-34 (1997); Lyons-Giordano et al., Exp. Cell Research186(1):39-46 (1990).

[0224] Sulfur-based modulators such as sulfonanilides and sulfonamidesmay also be used as modulators. Preferred sulfur-based modulatorsinclude sulfonanilide nonsteroidal anti-inflammatory drugs (NSAIDs) suchas nimesulide, acyclic sulfonamides, and malonyl alpha-mercaptoketonesand alpha-mercaptoalcohols. See, e.g., Bevilacqua et al., Drugs 46Suppl. 1:40-47 (1993); Hanessian et al., Bioorganic Medical ChemistryLetters 9(12):1691-96 (1999); Campbell et al., Bioorganic MedicalChemistry Letters 8(10):1157-62 (1998).

[0225] Another class of modulators includes compounds that antagonizereceptors involved in posterior segment ophthalmic disorders, e.g.,vascular endothelial growth factor (VEGF) receptors. VEGF antagonistsinclude peptides that inhibit the binding of VEGF to its receptors, suchas short disulfide-constrained peptides. See Fairbrother et al.,Biochemistry 37(51):17754-64 (1998); Binetruy-Tournaire et al., EMBO J.19(7): 1525-33 (2000). VEGF antagonists inhibit the outgrowth of bloodvessels by inhibiting the ability of VEGF to contact its receptors. Thismechanism of anti-angiogenesis operates differently than the mechanismcaused by the stimulation of growth factors such as bFGF, which act toinhibit angiogenesis by stimulating native inhibitors of proteases.Other VEGF antagonists may be derived from asymmetric variants of VEGFitself. See, e.g., Siemester et al., Proceedings of the National Academyof Sciences U.S.A. 95:4625-29 (1998). Other useful modulators are RNAaptamers, which may be designed to antagonize VEGF or the closelyrelated platelet-derived growth factor (PDGF), and may be administeredcoupled to polyethylene glycol or lipids. See, e.g., Floege et al.,American Journal of Pathology 154(1):169-79 (1999); Ostendorf et al., J.Clin. Invest. 104(7):913-23 (1999); Willis et al., Bioconjug. Chem.9(5):573-82 (1998).

[0226] Modulator screening may be performed by adding a putativemodulator test compound to a tissue or cell sample, and monitoring theeffect of the test compound on the function and/or expression ofoptineurin. A parallel sample which does not receive the test compoundis also monitored as a control. The treated and untreated cells are thencompared by any suitable phenotypic criteria, including but not limitedto microscopic analysis, viability testing, ability to replicate,histological examination, the level of a particular RNA or polypeptideassociated with the cells, the level of enzymatic activity expressed bythe cells or cell lysates, and the ability of the cells to interact withother cells or compounds. Differences between treated and untreatedcells indicates effects attributable to the test compound.

[0227] The invention thus also encompasses methods of screening foragents which inhibit promotion or expression of an optineurin moleculein vitro, comprising exposing a cell or tissue in which the optineurinmolecule is detectable in cultured cells to an agent in order todetermine whether the agent is capable of inhibiting production of theoptineurin molecule; and determining the level of optineurin molecule inthe exposed cells or tissue, where a decrease in the level of theoptineurin molecule after exposure of the cell line to the agent isindicative of inhibition of the optineurin molecule.

[0228] Alternatively, the screening method may include in vitroscreening of a cell or tissue in which an optineurin molecule isdetectable in cultured cells to an agent suspected of inhibitingproduction of the optineurin molecule; and determining the level of theoptineurin molecule in the cells or tissue, where a decrease in thelevel of optineurin molecule after exposure of the cells or tissue tothe agent is indicative of inhibition of optineurin molecule production.

[0229] The invention also encompasses in vivo methods of screening foragents which inhibit expression of the optineurin molecules, comprisingexposing a mammal having glaucomatous cells in which an optineurinmolecule is detectable to an agent suspected of inhibiting production ofthe optineurin molecule; and determining the level of optineurinmolecule in glaucomatous cells of the exposed mammal. A decrease in thelevel of optineurin molecule after exposure of the mammal to the agentis indicative of inhibition of marker nucleic acid expression.

[0230] Accordingly, the invention provides a method comprisingincubating a cell expressing the optineurin molecule with a testcompound and measuring the optineurin molecule level. The inventionfurther provides a method for quantitatively determining the level ofexpression of the optineurin molecule in a cell population, and a methodfor determining whether an agent is capable of increasing or decreasingthe level of expression of the optineurin molecule in a cell population.

[0231] The invention also encompasses a method for determining whetheran agent is capable of increasing or decreasing the level of expressionof the optineurin molecule in a cell population comprises the steps of(a) preparing cell extracts from control and agent-treated cellpopulations, (b) isolating the optineurin molecule from the cellextracts, (c) quantifying (e.g., in parallel) the amount of animmunocomplex formed between the optineurin molecule and an antibodyspecific to said optineurin molecule.

[0232] mRNA levels can be determined by Northern blot hybridization.mRNA levels can also be determined by methods involving PCR. Othersensitive methods for measuring mRNA, which can be used in highthroughput assays, e.g., a method using a DELFIA endpoint detection andquantification method, are described, e.g., in Webb and HurskainenJournal of Biomolecular Screening 1:119 (1996). Optineurin moleculelevels can be determined by immunoprecipitations or immunohistochemistryusing an antibody that specifically recognizes the protein productencoded by the nucleic acid molecules.

[0233] Agents that are identified as active in the drug screening assayare candidates to be tested for their capacity to block or promoteglaucoma.

[0234] H. In vivo Methods and Therapeutic Applications

[0235] The pharmaceutical compositions of the present invention,including antisense formulations, may be therapeutically used inclinical settings to affect glaucoma. As described above, the optineurinpromoter contains response elements which allow for the regulation ofoptineurin expression, and affecting the activity of a response elementcan at least partially inhibit or block glaucoma induced in cells byoptineurin expression.

[0236] As used herein, “at least partially inhibiting” refers to thereduction of a particular event, for example without limitation, thefunction and/or expression of optineurin polypeptides. In a preferredembodiment, to determine whether a particular event is “at leastpartially inhibited”, the sample of interest subject to a particularmethod or agent is compared with similar sample of interest notsubjected to the particular method or agent. In one embodiment, aninhibition of a particular event is statistically significant. In aparticularly preferred embodiment, a particular event is inhibited in asample of interest by 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90 %, 95% or100%, as compared to a similar sample of interest not subjected to theparticular event. More particularly, as used herein, “blocking” refersto inhibition of a particular event in a sample of interest by greaterthan 90%, as compared to a similar sample of interest not subject to theparticular event.

[0237] Accordingly, one aspect of the present invention is directed tothe use of optineurin nucleic acid molecules to at least partiallyinhibit, alter, or retard the development of glaucoma mediated byoptineurin. Another aspect of the present invention is directed to theuse of antisense optineurin nucleic acid molecules as therapeuticmolecules to at least partially inhibit or block (knockdown/knockout)expression of natural optineurin. A further aspect of the presentinvention is directed to the use of antisense optineurin nucleic acidmolecules as therapeutic molecules to at least partially enhance orincrease the expression of natural optineurin. The consequence ofaltering the expression of natural optineurin would be to affect theonset, progression, or development of glaucoma. A particular applicationwould be for the treatment of glaucomas, particularly those whereoptineurin is expressed at non-normal levels.

[0238] In yet another embodiment, a method for at least partiallyinhibiting the production of an optineurin polypeptide in a cell isprovided comprising: (a) providing an isolated nucleic acid moleculecomprising at least 10 consecutive nucleotides of the complement of SEQID NOs: 3 through 463; (b) introducing the nucleic acid molecule intothe cell; and (c) maintaining the cell under conditions permitting thebinding of the nucleic acid sequence to optineurin mRNA.

[0239] I. Markers

[0240] Another subset of the nucleic acid molecules of the inventionincludes nucleic acid molecules that are markers. As used herein, a“marker” is an indicator for the presence of at least one phenotype orpolymorphism, such as single nucleotide polymorphisms (SNPs), cleavableamplified polymorphic sequences (CAPs), amplified fragment lengthpolymorphisms (AFLPs), restriction fragment length polymorphisms(RFLPs), simple sequence repeats (SSRs), or random amplified polymorphicDNA (RAPDs). The markers can be used in a number of ways in the field ofmolecular genetics.

[0241] In one embodiment of the present invention, the markerspecifically hybridizes to a nucleic acid molecule having a nucleic acidsequence selected from the group of SEQ ID NOs: 1-463, fragments thereofand complements of either. In a preferred embodiment, the marker iscapable of detecting a SNP set forth in Table 2. In another preferredembodiment, the marker is capable of acting as a PCR primer to amplify aregion set forth in Table 1. Such markers include nucleic acid moleculesSEQ ID NOs: 1-463 or complements thereof or fragments of either that canact as markers and other nucleic acid molecules of the present inventionthat can act as markers.

[0242] Genetic markers of the invention include “dominant” or“codominant” markers. “Codominant markers” reveal the presence of two ormore alleles (two per diploid individual) at a locus. “Dominant markers”reveal the presence of only a single allele per locus. The presence ofthe dominant marker phenotype (e.g., a band of DNA) is an indicationthat one allele is in either the homozygous or heterozygous condition.The absence of the dominant marker phenotype (e.g., absence of a DNAband) is merely evidence that “some other” undefined allele is present.In the case of populations where individuals are predominantlyhomozygous and loci are predominately dimorphic, dominant and codominantmarkers can be equally valuable. As populations become more heterozygousand multi-allelic, codominant markers often become more informative ofthe genotype than dominant markers. Marker molecules can be, forexample, capable of detecting polymorphisms such as single nucleotidepolymorphisms (SNPs).

[0243] The genomes of animals and plants naturally undergo spontaneousmutation in the course of their continuing evolution. A “polymorphism”is a variation or difference in the sequence of the gene or its flankingregions that arises in some of the members of a species. The variantsequence and the “original” sequence co-exist in the species'population. In some instances, such co-existence is in stable orquasi-stable equilibrium.

[0244] A polymorphism is thus said to be “allelic,” in that, due to theexistence of the polymorphism, some members of a species may have theoriginal sequence (i.e., the original “allele”) whereas other membersmay have the variant sequence (i.e., the variant “allele”). In thesimplest case, only one variant sequence may exist and the polymorphismis thus said to be di-allelic. In other cases, the species' populationmay contain multiple alleles and the polymorphism is termed tri-allelic,etc. A single gene may have multiple different unrelated polymorphisms.For example, it may have a di-allelic polymorphism at one site and amulti-allelic polymorphism at another site.

[0245] The variation that defines the polymorphism may range from asingle nucleotide variation to the insertion or deletion of extendedregions within a gene. In some cases, the DNA sequence variations are inregions of the genome that are characterized by short tandem repeats(STRs) that include tandem di- or tri-nucleotide repeated motifs ofnucleotides. Polymorphisms characterized by such tandem repeats arereferred to as “variable number tandem repeat” (VNTR) polymorphisms.VNTRs have been used in identity analysis (EP 370719; U.S. Pat. Nos.5,075,217 and 5,175,082; WO 91/14003).

[0246] The detection of polymorphic sites in a sample of DNA may befacilitated through the use of nucleic acid amplification methods. Suchmethods specifically increase the concentration of polynucleotides thatspan the polymorphic site, or include that site and sequences locatedeither distal or proximal to it. Such amplified molecules can be readilydetected by gel electrophoresis or other means.

[0247] In an alternative embodiment, such polymorphisms can be detectedthrough the use of a marker nucleic acid molecule that is physicallylinked to such polymorphism(s). For this purpose, marker nucleic acidmolecules comprising a nucleotide sequence of a polynucleotide locatedwithin 1 mb of the polymorphism(s) and more preferably within 100 kb ofthe polymorphism(s) and most preferably within 10 kb of thepolymorphism(s) can be employed. Alternatively, marker nucleic acidmolecules comprising a nucleotide sequence of a polynucleotide locatedwithin 25 cM of the polymorphism(s) and more preferably within 15 cM ofthe polymorphism(s) and most preferably within 5 cM of thepolymorphism(s) can be employed.

[0248] The identification of a polymorphism can be determined in avariety of ways. By correlating the presence or absence of it in anorganism with the presence or absence of a phenotype, it is possible topredict the phenotype of that organism. If a polymorphism creates ordestroys a restriction endonuclease cleavage site, or if it results inthe loss or insertion of DNA (e.g., a VNTR polymorphism), it will alterthe size or profile of the DNA fragments that are generated by digestionwith that restriction endonuclease. As such, organisms that possess avariant sequence can be distinguished from those having the originalsequence by restriction fragment analysis. Polymorphisms that can beidentified in this manner are termed “restriction fragment lengthpolymorphisms” (RFLPs) (UK Patent Application 2135774; WO 90/13668; WO90/11369).

[0249] Polymorphisms can also be identified by Single StrandConformation Polymorphism (SSCP) analysis, random amplified polymorphicDNA (RAPD), and cleaveable amplified polymorphic sequences (CAPS). See,e.g., Lee et al., Anal. Biochem. 205:289-293 (1992); Sarkar et al.,Genoomics 13:441-443 (1992); Williams et al., Nucl. Acids Res.18:6531-6535 (1990); and Lyamichev et al., Science 260:778-783 (1993).It is understood that one or more of the nucleic acids of the invention,may be utilized as markers or probes to detect polymorphisms by SSCP,RAPD or CAPS analysis.

[0250] Polymorphisms may also be found using a DNA fingerprintingtechnique called amplified fragment length polymorphism (AFLP), which isbased on the selective PCR amplification of restriction fragments from atotal digest of genomic DNA to profile that DNA. Vos et al., NucleicAcids Res. 23:4407-4414 (1995). This method allows for the specificco-amplification of high numbers of restriction fragments, which can bevisualized by PCR without knowledge of the nucleic acid sequence. It isunderstood that one or more of the nucleic acids of the invention may beutilized as markers or probes to detect polymorphisms by AFLP analysisor for fingerprinting RNA.

[0251] Single Nucleotide Polymorphisms (SNPs) generally occur at greaterfrequency than other polymorphic markers and are spaced with a greateruniformity throughout a genome than other reported forms ofpolymorphism. The greater frequency and uniformity of SNPs means thatthere is greater probability that such a polymorphism will be found nearor in a genetic locus of interest than would be the case for otherpolymorphisms. SNPs are located in protein-coding regions and noncodingregions of a genome. Some of these SNPs may result in defective orvariant protein expression (e.g., as a result of mutations or defectivesplicing). Analysis (genotyping) of characterized SNPs can require onlya plus/minus assay rather than a lengthy measurement, permitting easierautomation.

[0252] SNPs can be characterized using any of a variety of methods. Suchmethods include the direct or indirect sequencing of the site, the useof restriction enzymes, enzymatic and chemical mismatch assays,allele-specific PCR, ligase chain reaction, single-strand conformationpolymorphism analysis, single base primer extension (U.S. Pat. Nos.6,004,744 and 5,888,819), solid-phase ELISA-based oligonucleotideligation assays, dideoxy fingerprinting, oligonucleotidefluorescence-quenching assays, 5′-nuclease allele-specific hybridizationTaqMan™ assay, template-directed dye-terminator incorporation (TDI)assay (Chen and Kwok, Nucl. Acids Res. 25:347-353, 1997),allele-specific molecular beacon assay (Tyagi et al., Nature Biotech.16: 49-53, 1998), PinPoint assay (Haff and Smirnov, Genome Res. 7:378-388, 1997), dCAPS analysis (Neff et al., Plant J. 14:387-392, 1998),pyrosequencing (Ronaghi et al., Analytical Biochemistry 267:65-71, 1999;WO 98/13523; WO 98/28440; and www.pyrosequencing.com), using massspectrometry, e.g. the Masscode™ system (WO 99/05319; WO 98/26095; WO98/12355; WO 97/33000; WO 97/27331; www.rapigene.com; and U.S. Pat. No.5,965,363), invasive cleavage of oligonucleotide probes, and using highdensity oligonucleotide arrays (Hacia et al., Nature Genetics22:164-167; www.affymetrix.com).

[0253] Polymorphisms may also be detected using allele-specificoligonucleotides (ASO), which, can be for example, used in combinationwith hybridization based technology including Southern, northern, anddot blot hybridizations, reverse dot blot hybridizations andhybridizations performed on microarray and related technology.

[0254] The stringency of hybridization for polymorphism detection ishighly dependent upon a variety of factors, including length of theallele-specific oligonucleotide, sequence composition, degree ofcomplementarity (i.e. presence or absence of base mismatches),concentration of salts and other factors such as formamide, andtemperature. These factors are important both during the hybridizationitself and during subsequent washes performed to remove targetpolynucleotide that is not specifically hybridized. In practice, theconditions of the final, most stringent wash are most critical. Inaddition, the amount of target polynucleotide that is able to hybridizeto the allele-specific oligonucleotide is also governed by such factorsas the concentration of both the ASO and the target polynucleotide, thepresence and concentration of factors that act to “tie up” watermolecules, so as to effectively concentrate the reagents (e.g., PEG,dextran, dextran sulfate, etc.), whether the nucleic acids areimmobilized or in solution, and the duration of hybridization andwashing steps.

[0255] Hybridizations are preferably performed below the meltingtemperature (T_(m)) of the ASO. The closer the hybridization and/orwashing step is to the T_(m), the higher the stringency. T_(m) for anoligonucleotide may be approximated, for example, according to thefollowing formula:

T_(m)=81.5+16.6×(log10[Na+])+0.41×(%G+C)−675/n;

[0256] where [Na+] is the molar salt concentration of Na+ or any othersuitable cation and n=number of bases in the oligonucleotide. Otherformulas for approximating T_(m) are available and are known to those ofordinary skill in the art.

[0257] Stringency is preferably adjusted so as to allow a given ASO todifferentially hybridize to a target polynucleotide of the correctallele and a target polynucleotide of the incorrect allele. Preferably,there will be at least a two-fold differential between the signalproduced by the ASO hybridizing to a target polynucleotide of thecorrect allele and the level of the signal produced by the ASOcross-hybridizing to a target polynucleotide of the incorrect allele(e.g., an ASO specific for a mutant allele cross-hybridizing to awild-type allele). In more preferred embodiments of the presentinvention, there is at least a five-fold signal differential. In highlypreferred embodiments of the present invention, there is at least anorder of magnitude signal differential between the ASO hybridizing to atarget polynucleotide of the correct allele and the level of the signalproduced by the ASO cross-hybridizing to a target polynucleotide of theincorrect allele. While certain methods for detecting polymorphisms aredescribed herein, other detection methodologies may be utilized.

[0258] The identification of a polymorphism in the optineurin gene, orflanking sequences up to about 7,500 bases from either end of the codingregion, can be determined in a variety of ways. By correlating thepresence or absence of glaucoma in an individual with the presence orabsence of a polymorphism in the optineurin gene or its flankingregions, it is possible to diagnose the predisposition (prognosis) of anasymptomatic patient to glaucoma or related diseases.

[0259] In accordance with this embodiment of the invention, a sample DNAis obtained from a patient. In a preferred embodiment, the DNA sample isobtained from the patient's blood, however, any source of DNA may beused. The DNA is subjected to restriction endonuclease digestion usingthe optineurin promoter or fragments thereof as a probe in accordancewith the above-described RFLP methods. By comparing the RFLP pattern ofthe optineurin gene obtained from normal and glaucomatous patients, onecan determine a patient's predisposition (prognosis) to glaucoma. Thepolymorphism obtained in this approach can then be cloned to identifythe mutation at the regulatory region of the gene which affects itsexpression level. Changes involving promoter interactions with otherregulatory proteins can be identified by, for example, gel shift assaysusing HTM cell extracts, fluid from the anterior chamber of the eye,serum, etc.

[0260] Several different classes of polymorphisms may be identifiedthrough such methods. Examples of such classes include polymorphisms innon-translated optineurin gene sequences, including the promoter orother regulatory regions, and polymorphisms in genes whose productsinteract with optineurin regulatory sequences.

EXAMPLE 1 IDENTIFICATION OF SNPs IN THE OPTINEURIN PROMOTER

[0261] To identify novel SNPs in the promoter region up to 5 kb upstreamof the transcription initiation site, genomic DNA from 23 individuals issequenced. The individuals include 7 normal subjects, 8 POAG patientswith increased intra-ocular tension, and 8 NTG patients. DNA from theseindividuals is sequenced over 5000 nucleotides. Between 3 and 5amplicons are required to sequence the optineurin promoter region over 5kb, which number depends on the number and nature of repetitivesequences and GC richness of the promoter. Each amplicon is sequenced onone or both strands to detect presence of the SNPs.

[0262] Amplifications are carried out using a “hot-start” procedure.Samples are processed through 35 cycles of denaturation (95° C. for 30s) and annealing (55-60° C. for 30 s), followed by one last step ofelongation (72° C. for 50 s). PCR products are diluted in 5 volumes ofQiagen PB buffer (Qiagen, Valencia, Calif.), transferred onto a WhatmanGF/C filter plate (Whatman Group, Ann Arbor Mich.), washed two timeswith an 80% ethanol 20 mM Tris pH 7.5, and eluted in 50 microliters ofwater. Samples are quantified using the PicoGreen reagent protocol(Molecular Probes, Eugene, Oreg.). A second PCR is performed on anApplied Biosystem Gene Amp PCR System 9700 (96 wells) or 9700 Viper (384wells)(Applied Biosystem, Foster City, Calif.) to incorporate thesequencing dyes using a protocol of 25 cycles of denaturation (95° C.for 10 s) and annealing (55° C. for 5 s), followed by one last step ofelongation (59° C. for 2 min). PCR products are purified by the ABIethanol-EDTA precipitation protocol, collected in a Beckman-CouterAllegra 6R centrifuge (Beckman-Coulter, Inc., Fullerton, Calif.) andresuspended in a 50% HiDi-formamide solution. Samples are run on anApplied Biosystems 3700 DNA Analyser automated sequencer.

[0263] Sequence data is analyzed with the Staden preGap4 and Gap4programs Griffen, Computer Analysis of Sequence, Part 1 (Humana Press,1994). Sequencing data and all patients' information is stored in a 4Ddatabase on a MacIntosh G4. Data is transferred from the 4D database toSUN computers using CAP AppleShare server software. Several SNPs areidentified in the promoter region and their allelic frequencies inpatients and controls are calculated (Table 4). Genotypic frequenciesmay also be calculated for identified SNPs (Table 5). TABLE 4 SNPs andAllelic Frequencies Allelic Frequency of Variant Number of POAG NTGNormal Location^(\) CN* Subjects Patients Patients (control) 391 a/g 273/10 (30%) 5/8 (62.5%) 3/9 (33%) 709 g/a 29 3/10 (30.0%) 1/10 (10.0%)0/8 (0%) 887 t/a 29 1/11 (9.1%) 0/10 (0%) 0/8 (0%)

[0264] TABLE 5 Genotypic Frequencies for an Optineurin Promoter SNP SNPLocation^(†) Genotypic Frequencies & CN* Subject Group aa ag gg 2606POAG Patients 1 (9.1) 9 (81.8%) 1 (9.1) a/g (n = 11) NTG Patients 2(18.2%) 7 (63.6%) 2 (18.2%) (n = 11) Normal (control) 1 (24.3%) 5(71.4%) 1 (24.3%) (n = 7)

EXAMPLE 2 VECTOR CONSTRUCTION

[0265] Expression vectors can be constructed for efficient expression ofan optineurin promoter construct (e.g., the optineurin promoter operablylinked to a heterologous nucleic acid, etc.) in mammalian cell lines.These expression vectors generally include the optineurin promoteroperably linked to a nucleic acid sequence. The vectors can also bedesigned to confer antibiotic or toxin resistance through expression ofresistance genes under control of a second promoter. Illustrativevectors include pcDNA3.1 and pMEP4 (Invitrogen, Carlsbad, Calif.).

[0266] For example, the CMV2 promoter is deleted from mammalian vectorpTracer CMV2 (Invitrogen) and replaced with a nucleic acid moleculehaving SEQ ID NO: 1 linked in an manner that facilitates expression ofthe green florescent protein (pTrOp). Chinese hamster ovary cells (CHO)are then transfected with either pTracer CMV2 or pTrOp using the methodset forth in Cameri et al., Nature Biotechnology 14: 315-319 (1996).Levels of green fluorescent protein are measured using the method setforth in Cameri et al., Nature Biotechnology 14: 315-319 (1996).

EXAMPLE 3 MODULATOR SCREENING

[0267] The transfected cell lines described in Example 2 containingeither pTracer CMV2 or pTrOp are grown in a cell medium described byMiller et al. J. Biol. Chem. 274 20465-20472 (1999) supplemented by atest compound. The level of green fluorescent protein is measured usingthe method set forth in Cameri et al., Nature Biotechnology 14: 315-319(1996) across a range of test compounds and effective concentrations inthe CHO cell lines containing either pTracer CMV2 or pTrOp.

[0268] All references, publications, and patents cited herein arespecifically incorporated by reference in a manner consistent with thisdisclosure. Reagents and compositions (e.g., nucleic acid molecule,amino acid molecules, vectors, host cells, antibodies, etc.) related tooptineurin can be made using methodologies known to those of skill inthe art or may be obtained from commercial suppliers.

1 463 1 5054 DNA Homo sapiens allele 391 single nucleotide polymorphism(SNP) 1 gtacacctag aagtggaatt gctgggtcat atcataactc tctgtttaactttccaagga 60 actcatcctc ggaatatttg gaaccagtga tgaactgaat caaactaaagctgagacaaa 120 gtccagacca aggtcaacca tagggcagat gattcatgca gcgaccacaccagtggcctc 180 acaggagcag gggcacaccc tttgctgcag cagtccccaa catttttgacaccaggaact 240 ggtttcatgg aagacaattt ttccatggat ggtggtgggt gggggggtggttttgggatg 300 aaatgggtcc acctcagatc atcaggcatt agagtctcat aagaagcacgcaacctagat 360 cccttgcatg ttctgttgac aatagggttc acgctcctat gaaaatctaatgcagctgct 420 gatctgacaa gaggcggagc ttaggccata atgctcaccc acccgttgctcacctcctgc 480 tgttcggtct agttcctgag aggccacagg ccagtactgg ttcaccacccggggtttggg 540 gacccctgct ttattggaca taattattag gtcgtgttct ttttggtggtgtttgtacag 600 ctctattgag gtataatcca catgccataa aattcacccc atttgtaaatgtatgattca 660 tggctttcaa ttacacttaa aaagttgtaa aaccatcatt acaattcaaatttagtatat 720 ttccatcatc ccccaaaaat cccctcgagt tcctttgcag ttcaaagccacccccaattt 780 caggcaacta ctggtctgat ttctgtcttt ttctactttc cttttctggacatttaatgt 840 atatggagtc atagcatatg tagtctttgg catctggggt agcaagtacgaatattagtc 900 taccacctca gatgcacata aaaatattac atatcttttc ttttcttttccttccttcct 960 tccttccctc cttcctttct ctctctactt ccttccttcc ctccttcttacctttcttcc 1020 ttctctctct ctctctcttt ctttttggac agagtctcac tccatggcccaggctggagt 1080 gcagtggcac catcttggct cagcgcaacc tttgactccc aggctcaagcaattctcctg 1140 cctcagcctc tcaagtagct gagattacag gcacgcacca ctactgcctggctaattttt 1200 atatttttag tagagatagg gtttcaccat gttagccagg ctggtcttgaactcctgacc 1260 tcaaacgatc ctcccaaagt gctgggatta caggcgtgag ccaccgccctgggcctcttt 1320 actttcttta aacccagttc tgcaggggtg tacggaaacc tattttcgggcaccactggg 1380 gtctggagag gggaggtctc cttccctacg gccatgcaaa actccaggagggcttttggt 1440 acccattgaa gtaagggcca tttatttttc agcccagcaa cattgccactgataccctca 1500 ttatcaaatg gttcttctag ggaacagtct ctgctgtttc caatgacaagcctgggcagc 1560 agaatctgcg ggaggttccc aaagtccagt aggtgcatcc caagagcttgctgtctgtct 1620 gggtgctgca gggactgagg cttgagtcct tgatgctcat aagaccaccatcccactcct 1680 cctcccaatc tgggggcggg ggagtcactc ctccctccaa ctgttgtgaaagcctccacc 1740 ccacccagct ctggctcttc ctccaggaca tctggggtag atcatggatgtattgagatc 1800 aggctttctc aaaagacaag aagaaaggct gtacttctaa gagctgttgccaggagtcca 1860 gccaacgctc ctgaaggtag gaagcccaaa gggactcgtt gctaactccaaacagaggag 1920 attggggtgg aaagggaaca caaggaacat caaacccaga ttaggatctcactaaaaacc 1980 ttcccacact gctctacatt tacccaccac aaaaccacat caacaaatcagctaagagca 2040 tgctattatt tcagtttttt cgctgcattt agattccatc tacccatggaagtgtgcagg 2100 aagatggagt caccaaacgg gatgatccag gctaagaaac agaaccggctctaacacaag 2160 caacagcaac aaacaccatg agccaggcgt tcttctaggt gttgaagacgtatttcctct 2220 ttaatcttct cagcatcctt aggtgagggc tgtgggtcca gaggccttatctaaaatttt 2280 tgggtggctg ggcaccgtgg ctcacacatg taatcccagc actttggaaggccgaggcag 2340 gtggatcacc cgaggtcagg agttcaatac caggctggtc aacatggcgaaacctcatca 2400 atacgaaaaa tgcaaaaatt agcttggtgt ggtggcacac gcctgtaatcccagccactt 2460 gggaggctga ggcaggagaa tcactcaaac ccaggaggtg gagattgcagtgagctgaga 2520 ttatgccact gcactccagc ctgggcaaca gagtgagact ccacctcaaaaaataaaata 2580 aaacctttgg ggcaagctct gctttagagt ccagaattct ggggattttcaaaaggctat 2640 tcaataaatg ggatttatat cacataacac cctgacactg tctgacgcagttctcctatc 2700 aactattcga ttttccttca caaaacaaat ttaaaaatca catcaagggatctaaataaa 2760 gactgtaaat agctttccat cagttgggtc tggtcagaaa agaggtttggtccttagaac 2820 tttctggatt tgggagtgta ctatactccc cattttacag ataaagggaatgaggaaggg 2880 taagatgaag taacttggtc aaggtcctac agctaagaag tggttgtcgggggagtgtgt 2940 gtgcatgtgt gtgtgcagtg cttcagggca ccccccaccc cgaccccaccactgagagca 3000 aggaatcagg agaaaacaac tttgactgct ttctgtacca gaaactcacctcgagcctcc 3060 cacaccaaag ccatgggcag cttgtgggtg accttcttct cttggctctgagtttcactg 3120 atgctcattt taattcactt tcatagtgtt gttttgttct cgtttttgtttttgcttgag 3180 acaaagtctc cctctcaccc aagctggagt gcagtgctgc aatcacagctcattgcagcc 3240 tctccctcct gggctcaagc gatcctcctg ccttgacctc ccaaagtgctgggattacag 3300 gtgtgagcca ccgtgcccca cctatagggt tttaaacagt aaaaggagcctagtgaagta 3360 cgacttaccg caggcacccc ttacaggccc cggggggacc cttttctgccgatcccaggg 3420 tacagctgtg acaccgtctt ttctgcctgg attatcccag tagataaacaaaaattagag 3480 atcgtcattc catttctctc tgtatatatt tttccaagcc cttttcatgaatgatcagtt 3540 atttcctgca ctgatttttt tttttttttt ttttttttga gacggagtctcactctgtca 3600 cccaggctgg agtgcagtgg catgatctcg gctcgctgca agctctgcctcccgggttca 3660 agcgattctt ctgccttagc ctcccgagta gctgggacta caggagagtaccatcatgcc 3720 cggctaattt ttgtattttt agtagagaca ggctttcacc atattggccaggctggtctc 3780 gaactccgga ccttgcgatc tgcctgcctt ggcctcccaa agtgctgggattacaggcgt 3840 gagccaccgc gcctggccct cctgcactga tataaaaaga atttttttaaattctctatt 3900 tctccccact cccaccccca ggctcactcc ttataaagca gcctctagcctcatctgccc 3960 ctcctacacc acaccaactc gagggcccgg aattgggtct ggggcagtcgctgacttgct 4020 tgcttctctg ccctgctctt ggggttagcc tcaggggcag ggttgagagtcaggcttggc 4080 caggcagcag gaggtccaga cagcgaagca gaatccttcg gagataccaggagagggcgc 4140 atctgccttt ttcctgtttc agattaggtt ttgttgttgt tgttttgttttttttctctc 4200 cctctctccc tccctccctc tctccctccc tctctccctc tctccgtccctccctctctc 4260 tctctctccc cctccctcct tccctccctc ccatccctgc agcgctacccgggtactctg 4320 gatgcacata gggcggctct cgctcctacc ttgtcatcct gctgtctaatccgggggcag 4380 cttccctcct ccacaccagc agaggctatt cttcagcaac aagaatagccgagcctattc 4440 gtccgcaaca agagcccaag aagcatcctg caggctttct gctttttgagtgtattttaa 4500 agcaaaaacg agtggaaagc tatgtatgct cagttaacta tgtctagatgttaacctttt 4560 ttcaaaaaac acagatggag gcctccctcc gaggatgcct ggcattctcctctttctgtg 4620 ggcggcagcg accccctgcg gctccagcct ccactacggg atctgcgggaagacacgggg 4680 aagacgaact ccgcacactg catttgatta atgatttatt ttgattaacgccgtcacagt 4740 gacgccttag agcagtccct gttcacccgg gtcccagcct cgaccccgcacggacagcga 4800 gggtgggtag ctgggggcgg acgcaggaaa gaggaggggc ggggccttggtcgggtgggg 4860 tatggaatgg gcagggtggg ggggatgggc ggggtatggg atgggcggggcccgggaaat 4920 tccccggcgc gggcagggag cggctggctg tcagctgagc cgcgctgggcggggtcgcca 4980 ggccgcgcat cagccctagg caccccagtc ccggctgccc cctccgccaccgccgccgcc 5040 cgccggcagg ttcc 5054 2 46951 DNA Homo sapiens allele 391single nucleotide polymorphism (SNP) 2 gtacacctag aagtggaatt gctgggtcatatcataactc tctgtttaac tttccaagga 60 actcatcctc ggaatatttg gaaccagtgatgaactgaat caaactaaag ctgagacaaa 120 gtccagacca aggtcaacca tagggcagatgattcatgca gcgaccacac cagtggcctc 180 acaggagcag gggcacaccc tttgctgcagcagtccccaa catttttgac accaggaact 240 ggtttcatgg aagacaattt ttccatggatggtggtgggt gggggggtgg ttttgggatg 300 aaatgggtcc acctcagatc atcaggcattagagtctcat aagaagcacg caacctagat 360 cccttgcatg ttctgttgac aatagggttcacgctcctat gaaaatctaa tgcagctgct 420 gatctgacaa gaggcggagc ttaggccataatgctcaccc acccgttgct cacctcctgc 480 tgttcggtct agttcctgag aggccacaggccagtactgg ttcaccaccc ggggtttggg 540 gacccctgct ttattggaca taattattaggtcgtgttct ttttggtggt gtttgtacag 600 ctctattgag gtataatcca catgccataaaattcacccc atttgtaaat gtatgattca 660 tggctttcaa ttacacttaa aaagttgtaaaaccatcatt acaattcaaa tttagtatat 720 ttccatcatc ccccaaaaat cccctcgagttcctttgcag ttcaaagcca cccccaattt 780 caggcaacta ctggtctgat ttctgtctttttctactttc cttttctgga catttaatgt 840 atatggagtc atagcatatg tagtctttggcatctggggt agcaagtacg aatattagtc 900 taccacctca gatgcacata aaaatattacatatcttttc ttttcttttc cttccttcct 960 tccttccctc cttcctttct ctctctacttccttccttcc ctccttctta cctttcttcc 1020 ttctctctct ctctctcttt ctttttggacagagtctcac tccatggccc aggctggagt 1080 gcagtggcac catcttggct cagcgcaacctttgactccc aggctcaagc aattctcctg 1140 cctcagcctc tcaagtagct gagattacaggcacgcacca ctactgcctg gctaattttt 1200 atatttttag tagagatagg gtttcaccatgttagccagg ctggtcttga actcctgacc 1260 tcaaacgatc ctcccaaagt gctgggattacaggcgtgag ccaccgccct gggcctcttt 1320 actttcttta aacccagttc tgcaggggtgtacggaaacc tattttcggg caccactggg 1380 gtctggagag gggaggtctc cttccctacggccatgcaaa actccaggag ggcttttggt 1440 acccattgaa gtaagggcca tttatttttcagcccagcaa cattgccact gataccctca 1500 ttatcaaatg gttcttctag ggaacagtctctgctgtttc caatgacaag cctgggcagc 1560 agaatctgcg ggaggttccc aaagtccagtaggtgcatcc caagagcttg ctgtctgtct 1620 gggtgctgca gggactgagg cttgagtccttgatgctcat aagaccacca tcccactcct 1680 cctcccaatc tgggggcggg ggagtcactcctccctccaa ctgttgtgaa agcctccacc 1740 ccacccagct ctggctcttc ctccaggacatctggggtag atcatggatg tattgagatc 1800 aggctttctc aaaagacaag aagaaaggctgtacttctaa gagctgttgc caggagtcca 1860 gccaacgctc ctgaaggtag gaagcccaaagggactcgtt gctaactcca aacagaggag 1920 attggggtgg aaagggaaca caaggaacatcaaacccaga ttaggatctc actaaaaacc 1980 ttcccacact gctctacatt tacccaccacaaaaccacat caacaaatca gctaagagca 2040 tgctattatt tcagtttttt cgctgcatttagattccatc tacccatgga agtgtgcagg 2100 aagatggagt caccaaacgg gatgatccaggctaagaaac agaaccggct ctaacacaag 2160 caacagcaac aaacaccatg agccaggcgttcttctaggt gttgaagacg tatttcctct 2220 ttaatcttct cagcatcctt aggtgagggctgtgggtcca gaggccttat ctaaaatttt 2280 tgggtggctg ggcaccgtgg ctcacacatgtaatcccagc actttggaag gccgaggcag 2340 gtggatcacc cgaggtcagg agttcaataccaggctggtc aacatggcga aacctcatca 2400 atacgaaaaa tgcaaaaatt agcttggtgtggtggcacac gcctgtaatc ccagccactt 2460 gggaggctga ggcaggagaa tcactcaaacccaggaggtg gagattgcag tgagctgaga 2520 ttatgccact gcactccagc ctgggcaacagagtgagact ccacctcaaa aaataaaata 2580 aaacctttgg ggcaagctct gctttagagtccagaattct ggggattttc aaaaggctat 2640 tcaataaatg ggatttatat cacataacaccctgacactg tctgacgcag ttctcctatc 2700 aactattcga ttttccttca caaaacaaatttaaaaatca catcaaggga tctaaataaa 2760 gactgtaaat agctttccat cagttgggtctggtcagaaa agaggtttgg tccttagaac 2820 tttctggatt tgggagtgta ctatactccccattttacag ataaagggaa tgaggaaggg 2880 taagatgaag taacttggtc aaggtcctacagctaagaag tggttgtcgg gggagtgtgt 2940 gtgcatgtgt gtgtgcagtg cttcagggcaccccccaccc cgaccccacc actgagagca 3000 aggaatcagg agaaaacaac tttgactgctttctgtacca gaaactcacc tcgagcctcc 3060 cacaccaaag ccatgggcag cttgtgggtgaccttcttct cttggctctg agtttcactg 3120 atgctcattt taattcactt tcatagtgttgttttgttct cgtttttgtt tttgcttgag 3180 acaaagtctc cctctcaccc aagctggagtgcagtgctgc aatcacagct cattgcagcc 3240 tctccctcct gggctcaagc gatcctcctgccttgacctc ccaaagtgct gggattacag 3300 gtgtgagcca ccgtgcccca cctatagggttttaaacagt aaaaggagcc tagtgaagta 3360 cgacttaccg caggcacccc ttacaggccccggggggacc cttttctgcc gatcccaggg 3420 tacagctgtg acaccgtctt ttctgcctggattatcccag tagataaaca aaaattagag 3480 atcgtcattc catttctctc tgtatatatttttccaagcc cttttcatga atgatcagtt 3540 atttcctgca ctgatttttt ttttttttttttttttttga gacggagtct cactctgtca 3600 cccaggctgg agtgcagtgg catgatctcggctcgctgca agctctgcct cccgggttca 3660 agcgattctt ctgccttagc ctcccgagtagctgggacta caggagagta ccatcatgcc 3720 cggctaattt ttgtattttt agtagagacaggctttcacc atattggcca ggctggtctc 3780 gaactccgga ccttgcgatc tgcctgccttggcctcccaa agtgctggga ttacaggcgt 3840 gagccaccgc gcctggccct cctgcactgatataaaaaga atttttttaa attctctatt 3900 tctccccact cccaccccca ggctcactccttataaagca gcctctagcc tcatctgccc 3960 ctcctacacc acaccaactc gagggcccggaattgggtct ggggcagtcg ctgacttgct 4020 tgcttctctg ccctgctctt ggggttagcctcaggggcag ggttgagagt caggcttggc 4080 caggcagcag gaggtccaga cagcgaagcagaatccttcg gagataccag gagagggcgc 4140 atctgccttt ttcctgtttc agattaggttttgttgttgt tgttttgttt tttttctctc 4200 cctctctccc tccctccctc tctccctccctctctccctc tctccgtccc tccctctctc 4260 tctctctccc cctccctcct tccctccctcccatccctgc agcgctaccc gggtactctg 4320 gatgcacata gggcggctct cgctcctaccttgtcatcct gctgtctaat ccgggggcag 4380 cttccctcct ccacaccagc agaggctattcttcagcaac aagaatagcc gagcctattc 4440 gtccgcaaca agagcccaag aagcatcctgcaggctttct gctttttgag tgtattttaa 4500 agcaaaaacg agtggaaagc tatgtatgctcagttaacta tgtctagatg ttaacctttt 4560 ttcaaaaaac acagatggag gcctccctccgaggatgcct ggcattctcc tctttctgtg 4620 ggcggcagcg accccctgcg gctccagcctccactacggg atctgcggga agacacgggg 4680 aagacgaact ccgcacactg catttgattaatgatttatt ttgattaacg ccgtcacagt 4740 gacgccttag agcagtccct gttcacccgggtcccagcct cgaccccgca cggacagcga 4800 gggtgggtag ctgggggcgg acgcaggaaagaggaggggc ggggccttgg tcgggtgggg 4860 tatggaatgg gcagggtggg ggggatgggcggggtatggg atgggcgggg cccgggaaat 4920 tccccggcgc gggcagggag cggctggctgtcagctgagc cgcgctgggc ggggtcgcca 4980 ggccgcgcat cagccctagg caccccagtcccggctgccc cctccgccac cgccgccgcc 5040 cgccggcagg ttccctggtc agcgtcccatcccggtcggg agttctctcc aggcggcacg 5100 atgccgagga aacagtgacc ctgagcgaagccaagccggg cggcaggtga gccagggcag 5160 ggggctgcag cggtgggcga gggggcggcggctccttccc ggcgggcgct tccgcggcct 5220 gaaaacggta cccgccgccc tgccccgcggcccggagcct gtcggggtga gggtggcgag 5280 gggtggggcg gccctcccgc gagacggctgccctgggagg atccccaagc ctcggcgggt 5340 cctgacctgg tgggagaggg tgggtagtgaacacagggtg ggcccaagaa gggtccaggg 5400 ccggcctctt ggccgcaggt aacccctctacaagtcaccc agagacttgg aggtgacgtc 5460 ctcccgcagg ctgggaagga ttccgcagccagttcctcgc cataaggagg tggttacaga 5520 cacgcctgtg ggagacatgg gaccactacccgagctgaat gaggtgccca gggtcggtgg 5580 gaatccacat agactgctgt gtctaggagagtgtgtacac tctatataga gagaatattt 5640 gatgaggccg agaaccccaa aggaattgtttttgccatcg aaatgcataa gctacaggaa 5700 gtaaacatcg ctcaaagaaa atgaaacccaggttccgacc tggcctccac cagcaggaaa 5760 actgttgtat ggaaatgtac accaaggaactgtttgtaag gatgaaatgg aaaatgaaag 5820 catagaaaag taaggcaatg tgaggacctcagaagccttt ggatctggtt agcaaggtgc 5880 aggctgtgtg aacagaagaa cccaccttgctcttttgacc cagggagaga agagttagtg 5940 gtgccaggct gcctcccagg acaggcccagttttggggcg ggggctggag aagtcccagg 6000 gcagacccca aggccaggga ttgccctggagtggacaagg caagtttaaa tgtgacctgt 6060 agacagagga cgatcattga tttaagtcctggcataattc atctgttttt ccactgggcc 6120 aattttaggc gttctttata tattacgtacaacacaataa aagtaacagc ttcttttttt 6180 ataaactaac ctttaggttg catattatgctaggaatata actaacctaa cctccacccc 6240 cccacccccc cacccacccc cggaaaaccaaactttattc tcgagggcaa ctggacactg 6300 tattatgtcc tgccgaaccc ccgcccttacttaggtagag gcgcataagt attctggcca 6360 tgaaatacac cttctgtttt atagagatttggcatatttg atatatgttc tttggaattt 6420 atgaaataga aaacccaaag gaaaaagaaaaaaattaaac ttttttttaa aaaaatattt 6480 catttagaaa gaaaaaatta aaagtttttgttatttccac tttgacatgt gtatgaggag 6540 cattagaatc tgtgttatga agtgatacccagatggcttt gcagtgacag aaaacacttc 6600 taatttttca aataagttag aaaggaggattttgagaatc tggaagtgaa tgccgtggaa 6660 ctgtggagaa actcctaaca atactggcatgctgagtttg tcacctacac agcagaaagc 6720 attttacagg tattagactt aaaaatacctcattatgcta tattttcatt tcatttcaac 6780 caacatttac tgcaagcctg ctatgtgccagtctctagta agaataacat ggagagaaaa 6840 tagacgggca gacgatagca gtaatttcagaatgggcgga ggaggtggaa atgcagagtg 6900 ttcttgggtg ctcggagtag ggaacagttcatatgacaca ggacatcata tttaagctgg 6960 acattgatct gtccagtgga agtttgccaggacacaaggg gagccagaca tttcgggcag 7020 agggcagaag ggaggcaagg acgcagagacagaagaatgc agggaaccgt gagctcttta 7080 acgtgccttg ggtacgagtg ttttggtggcaggaggtgac tctggtgggt cagttgggcc 7140 tgggccctga aaggccacgt ttgtcttgttaagaaactga aagtctgtcg tgtatgggaa 7200 gggtggactc aggggaatga cctcatattgcaaaagaaac catgagggca agaggactga 7260 aattgacagg gctgaaagcc agtcatttaggaggctgtta aaatgtattc aacagatatt 7320 taccgagtcc tgccttctat gtggcaagtaatgttctcag tcctggggat acagcagtgg 7380 acaaaatagg cagaacccct gtcctttgtggaggtcaaca tggctgtggt acagacaaga 7440 taagggcctg aactaagcct gtggggatggagaggaggca gtggatttta gaaacactta 7500 gaagggccag gcatggtggc tcatgcctgtaatcccagca cttagggagg ccgaggtggg 7560 tggatcactt gagatcagga gttcaagaccagcctggcca acgtggcaaa accctgtctc 7620 tactaaaaat acaaaaatta gctgggaatggtggcatgca cctgtaatcc cagctacttg 7680 ggaggctgag gtgggagaat tgcttgaacctgggaggcgg aggttgcagt gagctgagat 7740 tttgccacgg cactctagcc tgggcgacagagtgagactg agtcttaatt taaaaaaaaa 7800 aagaaaaaat aaatatttag gaggtagaggtgatggcatt tggtgaccca ttagacttgg 7860 ggaaaagggg gctgggaaga atcaagccttactcccaggt tcctgccttg gggagctagg 7920 ttaaagctgc tgccattttc tgtatattactaaaatacga agtcacaata tcccttctag 7980 tgtgtaatga cgaggctgct tttaccccattagtaggatg catactctaa gttctttttc 8040 caaggtggga agggtttagt caatgcaatgttagaatctt accccccttg tgaacccaaa 8100 ctcttcatag catttgccag tagccaactcttgtctctgg tcaaaaagca agttttcact 8160 gtttaaaact catttatccc atgtagggattgaacctacg aatacttcat tagcaaattt 8220 gaagacttgg ccataccagg gcaagggcctaaccttatac tatgtgcagt gtccactggg 8280 gggaagtcag tgtaaggcag gtaccatttgacacattatg actttctgac cactagagcc 8340 atcccacagt ggaacagaca gtgcctgctaaggtcactgc actcgtgctc tgtcattgaa 8400 atactcaagc ctgctctata ggatcatgtgccatcgaaac cagacaagac attcttgcat 8460 tgaatatatt ggtctggatc atttctaaggccaactcagc aatcagatgt atgctcgtgt 8520 ttacaaggtc aggtcattac tagaattacttgatctctgg aatttaaatg tatatacaca 8580 cacatgcaca cacacacaca cacacacacacaatcaggaa ttgagatagc agatctggga 8640 ataaaatcta ggggacttgc ttttggccatccatctacta ggcagtcctc agtcttcctc 8700 attggtggaa tcttgacaaa tcctgtgctcaattaattca tccttccatc attaaatatc 8760 acttttgcct agaaataaca tacttcacatctaggtcagt tgattatggg aaactctgtt 8820 atttgataga atcttagaat aaatggcttagttccaattt ctgtaacctt cgaagacatg 8880 tgcaaggata tgtaatgtat ctctgtctttcctcagttga atgtagctca aggtgatgtt 8940 aaaaacagag aatgaagctt tctgtaactggcattccacc gacagatatt tcatatgtgc 9000 actgcacagt gatagctcat agtgatgtcctaagataacc taagaattat gaagtgccct 9060 ttgagtcatc caaaatcagt taaattagtttttttttctt tttttttttt tttttttttg 9120 tgagacaggg tctcactctg tcacccaggctggagtgcag tagcatgatc ttagctcact 9180 gcaacctctg cctcccaggt tcaagcgattctcatgcctc agctcccgag tagctgggac 9240 tacaggcatg caccactaca cccagctacttttttttttt ttttttttta ctattacaaa 9300 atttatttaa caaaaagtct aatatgaaaatgtacatgac ctaactttta catcatagta 9360 aaacaggccc tatggagaga ggacatgggtttctctgctg aacagccatt ttttatactc 9420 attccaaggc ttctaacatg aggatactgtttcctcgtat taccaccatt ccagtattgt 9480 tctgttgccc actagtcgcc atctccacacattcatctat cacaagattc ataaagggat 9540 caagtccctg caatattcct tggacgtgtctgccaccatt taatttcaat gataacttct 9600 tgtccataaa tcccttctag ccatctggttagaacatgat gctattcgag atgttcaaca 9660 aaaggtggca ttcgagatct tcaaggaacgaagaagggac agcatggggg tgaccagggg 9720 cccgaccgca ggaccgggag gcgagtcggccagaaagagg tgcagtggct gctggtggta 9780 actacgccga cgtgcgagct ctgctactaatttatgtatt tttagtagag atgggatttc 9840 accatgttgg ctgggctgat ctcaaactcctggcctcaaa tgatctgccc accttggctt 9900 cccaaagtgc tgggattaca ggcataagccactgcacccg gcatcaatta aattactttt 9960 agtaaaattt tggtattaag tatttacatgcttatattta ccgtttatgc taacttctta 10020 tgtttgatga catttgaatt acaaaatattttttctaaca atacacaata tcacagaatt 10080 tgctgtcatt aggacagtta tagctattcatgagagtgac tggcctgccc gtgatacttg 10140 gtgtatatgc attgaagggt ggggtttagattgtggtctt tctagaaact aaagaatatt 10200 tttcacagag ttcataagga ttatcatttgctggatgagt caataaatac cactagaggt 10260 catgtgactt cccaaggcca cacatctggctaatggtaca aatgtttttt tctgtggcct 10320 cccttagcac gaccccactt ttttcctcgtatgcttataa aatccgggca atgtgagaag 10380 tcagaaacac tgatgtaaca actagaaccagttttaactt tattatggtg gtatctgtag 10440 gcttggtgga ggcataaaag gtcatggaggggcccggcac cgtggctcaa gcctgtaatt 10500 ccagcacttt gggaggctga gatgggtggatcacctgagg tcacgagttc aagaccagac 10560 tggccaactt ggcaaaaccc tgtctctactaaaaatacaa aaattagccg ggtgtggtag 10620 cacatgcctg tgatcccagc tacttgggaggttgaggcag gagaatcact tgaatctgaa 10680 aggcagaggt tgcagtgaac tgagattgtgccactgtact ccagcctggg cgacaaagtg 10740 agactctgtc tgaaaaaaaa aaaaaagttcattgaggaaa aaaatcttaa cagttttcaa 10800 agaaacgtct ctctcttttc ttccctctcttctcccctcc ccttcctttc attttcagaa 10860 aaaaaggaga aatttgtgca tataataagatgaaaccaaa cacacttcta aaaatctgct 10920 tagatccttt ctttaaaaaa atctgttgaatattttcatg gcctttgcaa aatatagtat 10980 aatcactgaa tacctaagaa aaattaactgttggcaagta aatgatacag atgacaagga 11040 ttttctcttt taggtctatt acctagaattttagtattcc tctgcaaatt aggtaggaac 11100 actgttactc agtaaaaccc tattttaatgagatgattct gggtacaaaa aaatgaattt 11160 tacagaaaat tagaaactga tggtctattcagattatttt tgtgagaaga agagcagtct 11220 gttctagtca cctaatgcta agctatggaacacttctgca ttatacctgt tttctaagtg 11280 aatttgggtg tgtgacacat agatacaaaaagttcaaaaa tgaatcctat ggtttatcag 11340 tgttttctgc ttcgtaagat tgccatcaccctcattacat gactacatga gaatgcccct 11400 cttggtacta gctcttgtag gaagaagtggcacaggctgt actgtcacag ggtgtggtag 11460 gtagctctgt ctctgattac ctacaagttcctcacaccaa aaggaggtta ccaggtattg 11520 cttccagaag atctacagaa agcccaaggtattgcctcct gtggccaagg acgcaacacc 11580 acagtggtgc attttgtttt attttaattgatggctatta aatgcacaca tagatatcat 11640 gacacagtta catgtcagaa acaggcagtgctcttatctc tatcttccag ggactgatat 11700 catgtgtaat ggattcttca caactcagggttcacacagg ctccatagtc atgagtggct 11760 gatggatctc attgagacca gtgcctttcccccgttagaa atgggatgct cgcttccaag 11820 ttctctcctc accccttttg tgcatgcgtggctttctgcc tgatcacagt tgtatagtgt 11880 acattggtgc ttaataccta tttattggatagatggattt taaattattt tctaaggtcc 11940 aggagcagtg gcttatgcct gtaatcccagcacttttgga ggccgaggcc agcagatcac 12000 ctgaagtcag gagttcgaga ccggcctggctaacatgatg aaaccccgtc tctactaaaa 12060 atacaaaaat tggccaggca tggtggctgacgcctatagt cccagctact tgggaggctg 12120 agtcaggaga atcacttgaa cccaggaggtggaggttgca gtgagccgag atcgctccac 12180 tgcactccag cctgggcaac agagagagactctcaaaaaa ccaaaacagt cttttttttt 12240 tttttttttt gagactctgt cgcccatgctggagtgcagt ggcacgatct cggctcactg 12300 caagctctgc ctcctgggtt cacgccattctcctgccgca gcctccggag tagctgggac 12360 tacaggcacc tgccaccatg cttggctaattttttgtatt ttttttatta gagacagggt 12420 ttcaccgtgt tagccaggat ggtctcgatctcctgacctc gtaatctgcc cgcctcagcc 12480 tcccaaagtg ctgggattag aggcgtgagccaccgcgccc ggctcaaaaa caatcttcta 12540 acagtaaaca tcctggaagt aaataatatgctttttcagg catgtctctg gactttggcc 12600 attcttgacc tttgtagaat gcgtgtgtgggccatgtgta cacagcgtca gcggggagga 12660 gttgcctttg ctatgtattg ctttataatctatccccatc agctcttgaa gaaaatacat 12720 cattttcacc aagtgtgaag gtggaaatttgtgctgaaac atgtttgcct gggtggttgg 12780 taacacagaa gcagagtggg gatttactcattggtcttgc atttctatgt ccacatggat 12840 gcctctacaa aacaaaatga tatgtgtaaaaaaatttcag accatgcaat acctaaagat 12900 atcttagtct tttcagtatg cattgaaaaatagactatta aagcctaagt actcagtaat 12960 ataactttgt tgttttacaa ggtgtggctttgatagctgg tggtgccact tcctggcctt 13020 ggatgagccg tacgcctctg taaacccaacttcctcacct ttgaaacagc tgcctggttc 13080 agcattaatg aagattagtc agtgacaggcctggtgtgct gagtccgcac atagtaagca 13140 ttcaaagaat gttaattctc cctttcttcttaaccaaaaa cacaataaac taaatatagg 13200 ttttaaaaca gcttttttga gatagaattcacttaccatg caatttactt gaaataagga 13260 gatttgaatt tgaagatttg catgggaaatttttttcatg ataccttgcc catgtctaca 13320 tatatcctga tcatcatgat gtcataccctcgtctccctc ccccatttcc caaatcctta 13380 ttgtaccctt tttttttttt tttggtttgagacggagtct cgctctgttg cccaggctgg 13440 agtgcagtgg cgcgatcttg gctcactgcaagctctgcct cccgggttca cgccattctc 13500 ctgcctcagc ctcccgagta gctgggactacaagcgccca acaccaagcc cggctaattt 13560 tttgtatttt tagtagagac ggggtttcactgtgttagcc aggatggtct caatctcctg 13620 acctcatgat ctgtccgcct cggcctcccaaagtgctggg attacaggcg tgagccacca 13680 cgcccggccc tcattgtacc cttttatacacccatacaca cacacgcaca cacacacatg 13740 cacacatgcg cgtgcacaca cacacacacttttctgaagc tacatatacc ttttttgttt 13800 aaaaggaaga atcaaaaatg tccaaaatgtaactggagag aaagtgggca acttttggag 13860 taagtattag caatcgccaa tgggtttgtgggactcccgg ggaccccttg tggggcgggg 13920 gacagctcta ttttcaacag gtgacttttccacaggaact tctgcaatgt cccatcaacc 13980 tctcagctgc ctcactgaaa aggaggacagccccagtgaa agcacaggaa atggaccccc 14040 ccacctggcc cacccaaacc tggacacgtttaccccggag gagctgctgc agcagatgaa 14100 agagctcctg accgagaacc accagctgaaaggtgagcag ggctggcccc tgtgtgcccc 14160 attcatcctg ggcctgcaag aaatgccatccctttgcact aaggcttggt ggtgagctcc 14220 cttctccccg tttccatagg tggtagctggtggggaagca caggatttag catttggcaa 14280 ggctaaatct gttctgattt ttacttttggaaacaggtac aagtaaaaac tgtgtgtatc 14340 tcaaggaagt agcataatga tatttagcccattcaaaagg aaaaagaggc tgggcgtggt 14400 ggctcatgcc tgtcattcca tcactttgggaggccgaggc agaaggattg cttgagtaca 14460 ggagttcaag accagcctgg gcaagatggcaagacctgat ctctacaaaa aaattaaaaa 14520 aaaaaaaaaa aagctgggcg tggtggtgcacgcctctggt cctagctact ggggatgctg 14580 aggttggagg attgcttgag cctgggaagttggagctgca gtgagccatg atcgtgccac 14640 tgcactttag cctggatgac agagagagaccctgactcaa aaaaaaaaaa aaaaaaagga 14700 aaaaggaaga aaggctgcta tggttccagagttagtccta tatattacct tattaagaga 14760 aagcatcctg gtatctcaag atggctttgggcaggaccag tatttgaatc taggagtagt 14820 aagaacttcc ttagctccta gtaaccatagatatttagat atttgtgctg tagtggcggt 14880 acccaaatcc actttatttt cttgggatttttaaggacta gaaatgatgt tcatcccgct 14940 agtcttttct gtaagcaaaa accacttcgtctttttgctg ctgacccttg ggccaaggct 15000 aagcatggca tctttcaatt cagagccatgtggtcaagtg gactagaggg agatttggtt 15060 catcagatca agtccacttt cctggtgtgtgactccatca ctctgaacct cctgcagaag 15120 ccatgaagct aaataatcaa gccatgaaagggagatttga ggagctttcg gcctggacag 15180 agaaacagaa ggaagaacgc cagttttttgagatacagag caaagaagca aaagagcgtc 15240 taatggcctt gagtcatgag aatgagaaattgaaggaaga gcttggaaaa ctaaaaggga 15300 aatcagaaag gtcatctgag gtgagcagaccgatccattg tgatgttgtt tttttttttt 15360 cccttgacat ttgcagtgga atcttacgtgtctagactcc tagatcaaaa cctttcatgg 15420 ttcagtctgg attggtgttt tgcctggtcttggaagaagt gcttttgctg aaaagattgg 15480 ttgccctatt aagggtcatg gataatctcttttagaagaa agaaatttgt aaagctttga 15540 ccgtactgat tgtaggcaaa agaacagtaaggttataaat cattgtattg tattcattat 15600 agatggtgca gatgggcctc tgcctagaaccaacaattgt ttttagtttg tctttgatat 15660 aaaaaatatg tttaaaaaac ccattactcagaatttttac ttgttgacct tgtctgttct 15720 ctcagtctaa aatggagatt attcactttacattttcctt tttaaaaatg ctttggaaaa 15780 tgtcatgttg tggtaggagg ctatcgcattgccacagatg aaaagagaaa gagacacatt 15840 tttcttaacc caaagaacct ggaaaaatgtgctcatacct gggagtggat gtcaaagatg 15900 atagtaatga cacaacccag aacaagtttgaaatccccac tgggcgtggt ttgttaagga 15960 gtcctctgct gtccatgcca agtgtggggagaatgggtgt gggtgggctt tgatgggagg 16020 aaaagggcag gaggttgggt ggtgggaacgttttttcctt cctttctgga attttagaca 16080 cagcttatga gtccactgtt gccaaagtgtggttgattat ttctatgata tcagattatt 16140 ccagcatggc aaggaaggct ttctttcttgtcatgaagtt actttaaatt ttgattattt 16200 gaatacaata aaataatgta aaaatttccattttaaatgt tatgtcaaaa taaatcgtgg 16260 gctaggcacg gtggctcaca cctgtaatcccagcgctttg ggaggctagg gtgggcagat 16320 cacctgaggt gaggagttcg agaccagcctggccaacatg gcgaaacccc atctctacta 16380 aaaatacaaa aattagccag gcatggtagtgcgtgcctgt agtcccagct acttgggagg 16440 ctaaggcagg agaattgctt aaacccgagaggtggaggat gcagtaagct gagatcgcgc 16500 cattgcactc caatgggcga cagagtgagactctgtctgg gaaaaaaaaa ttgtcaggta 16560 aaagctgaaa ttttctacat taaagcacaaggcataaagg tgttgagaaa cttccttgtc 16620 agtaggtgtg ggggcaattg agtctcatggccaggtcctt agtttgattt gtatttgttt 16680 tttgactgtt tttttttttt tttttttttgagatggagtc ttgctctgtt gcccaggctg 16740 gagtacagtg gcataatctc ggctcactgcaagctccgcc tcccaggttc acgccattgt 16800 cctgcctcag cctcccgagt agctgggactacaggcgccc gccaccacgc ccagctaatc 16860 tttttgtatt tttagtagag acggggtttcactgtgttag ccaggatggt ctccatctcc 16920 tgaccttcat gatccgccca cctcggcctcccaaagtgct gggattacag gcgtgagcca 16980 ccacgcctgg cttggctttt ttttttttttttttgagaca gggtcttggc agtcttaaac 17040 tcctgggctc aggcagtctt cctgcctcagcctcccaact aatggggact acaggtgtgt 17100 gccactacac ctggctaatt attaaattttttgtaaagat gggggtcttg ctatgttgcc 17160 caggctggtc tcaaaatcct ggcctcaagggatcctccca cttcagcctc ccagagctct 17220 gcgattaagg gcatgagccc atggtgcccagccttagttt gatctgttca ttcactttac 17280 tccttgtcat ctccaggacc ccactgatgactccaggctt cccagggccg aagcggagca 17340 ggaaaaggac cagctcagga cccaggtggtgaggctacaa gcagagaagg cagacctgtt 17400 gggcatcgtg tctgaactgc agctcaagctgaactccagc ggctcctcag aagattcctt 17460 tgttgaaatt aggatggctg tgagtttttggttttatttt tgttttgagc aaactataaa 17520 gcctcccctg gaaagatgaa acaaataccactttttcttg tcaacacaag ccaaggattg 17580 aggaaattcc agtgtagcaa agataaattggctctcattt tctaagtata gcataatgca 17640 tgtaagggtt atcatagcta aaatggaaaaatattaatta ccttttatga tgaaagctgt 17700 agtctttttt tttttcttca tcatgtcctggcaaattgaa catttttgtg accagaaaag 17760 gaaaaaaccc acacgaacat gaactttctgtcatttttca aactaggtct caaagctgta 17820 ttccgcagtt cacttaaggg agcgcaaacatattttcaca acagaaccct ctttttttgt 17880 tttgagacag agtcttactc tgtcttcccggctggaatgc agtgatgtga tctcggctca 17940 ctgcaccctc tgcctccggg gttcaagagattctcgtgcc tcaacctccc aagtcgctgg 18000 gactacaagc gcatgccatc acacccggctaactttttgt atttttaata aaaaagacag 18060 gtttttgcca tgttggccag gctagtctcaaactcctggc ttcaagtgat ccacccgcct 18120 cggcctccca aagtgctggg atgacaggcgtgagccaccg cgcctggcca acagaacctt 18180 cttttcaaac aaagtggtat gaggaaccctgatacattaa aaagaagaag aggagaaaag 18240 aaagagcaga actgctctgg ttgtaggttgagggagtgtc ctggcttttc cttccctttc 18300 aaaagcagct actcaggagc ctctgagaactgagtttgaa gccattgcta tcaaaatcaa 18360 atttctctgc aaccccagat gaagtgggctaagcgagggg gccctaagct cttgagaagc 18420 atctgttaca actgtgcctg ggcataggggcagccctatt gaagagcaga gcaggtcgat 18480 gcaccagctg ggggcctgtc cttcattgctactaacaaag attagacagg gagaaagata 18540 gacaaggata aaatcctctg tagtatagatggtcactttc gatgagtcag agtacatatc 18600 tgatacagga aaagggactg gccgggtgtaatggctcacg cctataatcc cagcactttg 18660 ggaggctgag gaggcaggat cccttgagcccaggagttca agaccagcct gggcaacctg 18720 gcgaaaccct gtgtgtacat aaaaaaaaaatacaaaaagt tagctaggca cattggcaca 18780 cacctgtact cccagctact caggaggctgaggcaggagg atcacttgaa cctgggagtt 18840 tgagatgctg cagtgtgcca tgattgtgccgctgcactcc agcctgggtg acagagcaag 18900 actctgtctt taaatttaaa aaaaaaagaaaagaaaaaaa attgtgtcct tggaagagaa 18960 aaatgtacat tgtagataag tcaggaggagtggggaacaa cttgcaaaaa agctcaccta 19020 ctggttatat ctgaaatatg aatgtctgactgtctttgct ttctgattta tttgctgcaa 19080 tagagttagg aaacagctct gaataaccctgccatccatt ccccctcata catttcagtg 19140 gccaaaatca agataattaa aatgtcaattgaaaagcgta ttttgccaga gtaccccttc 19200 tgcaagtgat cgaagattac tgagacagaagtttaaccaa agaaacttac ccttctgtca 19260 ataaccgata agctgcagga aacccaacagcatatgagaa gtttccagat gaagcttctt 19320 ccttcagggt gcatggttag catctcattctccctgtcag atgagtgaca gttattttta 19380 gtgtaaacac gttttgcatg cgtttcttctgtgaactgga aagcactgcc caagatttag 19440 gaagaaacat caaaaatatc tagaacccccatggccccaa gcagagaaaa aatatatgta 19500 tctatgtttt ttataataga tatatctgtatttatattat ataatgaata tataatatat 19560 tctataatat ataatgtaga atatattctacaattatata attgcatata taattatata 19620 attatataat tatataatta tatatacatatatataatta tataattata taattatata 19680 attatataat tgtataatat attctacaattatataattg tattatatac acttacatat 19740 gtatctgtat tttttataat agatatatctatatttatat atatttatat atagatatat 19800 ctatatttat atatatttat atatagatatatctatattt atatatattt atatatatag 19860 atatatctat atttatatat ttttatatatagatatatct atatgtatat atatttatat 19920 atatagatat atctatttta tatatttttatatatattat atatatatat aaatttataa 19980 tatagatata tctatatatt tatatatagatatatctata ttttatatat atatttatag 20040 atatatctat atttatatat atatttatatatagatatat ctatatatat ctatatttat 20100 atctatattt atataatgaa tacatataatatattctata atatataata tataatataa 20160 catagaatat agaatatatt atacaattatataattgtat aatatatata cacttatata 20220 tgtatctgtt ttataataga tatatctgtatttatattat agaatatata taatatagaa 20280 tatatataat atattctata ttatataatatagaatatat ataatatatt ctatattata 20340 taatatagaa tatatataat atattctataatatataata tagaatatat tatacattat 20400 atattataca attaatatat aattacatatataacattat atatttatat aatatatatt 20460 tctatatcta aatatctaaa tatattatatatatatttat acgtatatac ctagaactat 20520 ctttgtgcat ttttgaagat tttccctgggagcttattgg aatataaatg tctttcaaat 20580 cctgtgggtc ttagactatc ttgttccctaagtgacctgt ggtgcataca aatttctaat 20640 gggaaccaac ttggccaaga tggtgctttgtgaatctcat tcacagaaac tgcctctttt 20700 ttaactttac ctcagtgagt tctagcattttgcattttaa aggaaggata tgtggagttg 20760 tcaccagctc tgtatgacct taaccttgagaaagagggaa ctgccaagga aagggaggag 20820 cagataagct ttcatgttta cagagtcaggtagaatgtgt atggcgagat gaaactgacc 20880 ttcacgcctt agctgggata tttataatcccgacagggcg tgccaggtga ggggagggta 20940 cgtttccatt tcctctgagc caccccgtttaaacagtgca catctgaatg tttggaagct 21000 tccttgggtt gcatgtcaca aaaattcatcttttgtcttt ttcttctttt gacaaagaat 21060 ttgtcttgta gacatattgt gttaaatcccttgcatttct gttttcacag gaaggagaag 21120 cagaagggtc agtaaaagaa atcaagcatagtcctgggcc cacgagaaca gtctccactg 21180 gcacgtatgt gaaggaagac tcgggctgtcaggcagacag gctgggcagg ctcgtcactg 21240 ggtgcttgtc accggaggtc aaatgttgtgacctgaggaa gtaacttctt tatgatttat 21300 accaggatct ttccagaata tttggtttgaatgctattta atgttgcagc tcaaactggc 21360 aaagattaaa aactgtttgg ttcctgtttggctcacactg actgctctgt tctagtggtg 21420 tctcacctcc agcagatgaa aagtgaaagcaaactggttc tcaatcaagt caatgatttg 21480 ttcctaatca aagacatgtt tgctcattggttccccggtg ccatttgacc cagaccagcc 21540 tgcccagctt ccataagtga aatattttcattttcttttc cctgctactt cccagttata 21600 agctggcatg gccaatactg gaacatcttttgtaacaatg actgatagca ctctcagtca 21660 ttgtgggtgt tgcctgaaag tgcccagatttcttatctgt ggagtctcaa gtgtacctgt 21720 cctatgtaga tgtgaggaaa cagacatcttaaatagtggc agggccttgg ggagggaggc 21780 agacctagaa ctgagcgcct gaacctcttgactctcgcaa agcagtgctc accaaggaga 21840 ctgctagctc gccttctgca agctgcttactcctgtaatc atctattcaa gagacgattg 21900 tctgaaaaga actctaagga tctctttttttttttttttt ttgagacgga atcttgttct 21960 gttgcccagg ctggagtgca gtggcgtgatctcggctcac tgcaagctcc gcctcccggg 22020 ttcacgccat tctcctgcct tagcctcccgagtagctgga accacaggcg cccaccacca 22080 tgcccggcta attttttgta tttttagtagagatggggtt tcaccgtgtt agccaggatg 22140 gtctcgatct cctgacctcg tgatctgcccgccttggcct cccaaagcgc tgggattaca 22200 ggcatgagcc accgcgcccg gccaggatctcttatctcac agacggataa gagatccatc 22260 tttttttttt ttttgagatg gagttttttttttttttttt tttttttgag atggagtttc 22320 actctgccac tgaggctgga gtgaagtggcgcaatctcag ctcactgcaa cgtctgcctc 22380 ccgggtacaa gcagttctcc tgcctcagccttccgagtag ctgggaatac aggtggccgc 22440 caccacgccc agctaacttt ttgtgtttttaatagagaca gggtttcact gtgttgacca 22500 ggctggtctc gaactcctga cctcaggtgatcctctggtc tcagcctccc aaagcgctgg 22560 gattacaggc gtgagccact gcgcccagctatctcataga tttgtaaaac cttctttgta 22620 taacttgatg aatgtgcata tagaatgacttacaaaacgt gaaaaaaatt gtcttccgtt 22680 atgtttctaa gcccttgtat aaggaagaaagtaagtatta gataatattc tttcgtcaaa 22740 cacagtaatt ggcataaacg aaagtaattcccttttttgg ttaacaattc ttcacgtttc 22800 ccccaaattg cttttgtcat atttaaagacgttcctggaa gtagtgggaa ataaaaaagc 22860 cctggttgaa atatcaaagc cacttcctcatcattcatat ccagatgcaa agaccgcttc 22920 ctcttttaag ccacatggct atttttaaaataacagctct gtaccatttg tgagtatcgt 22980 aattagtttg agattgattt ccaggtttggagttgaagct tcagagtcct ggaaaccggg 23040 atttaatcct ggctgtttat tagctatgcgaacctgagtc ggtgactgaa agagtagctg 23100 ccgttagcat catcatagtc atccctcatgttttgtaaca gtgatcatga ttctgtttgt 23160 cactgatatg tctcttgatt tagtcagattttgaaatcga gaaaaatact tgacatatca 23220 acagagcctc catttctctg ctctcattatttgaaaccac aagtgaaaaa ggttttctcc 23280 ccttgactta agctgtgatg gtctctgttaacttggagaa aggccagtgg tctgtacaat 23340 gtgcctttat cttttgtctg actgcagtcccctttgagac tagatctctg gaaagcttgg 23400 caccttcagc cacggctgcc tctgctgaactgttccgtga gttttgtggt gtggtgtgag 23460 gtacacagtg actgtttgga ggacgtgggtgtgtgcattg taagctggcc tctccagagc 23520 ctcactgagt ctccacacct tccctaggaagcatggagga gcttggcact gggggtccca 23580 ggaccagctg tgcttgttca ctagttgagaattagttgga gaatgttctg gaaagcagtt 23640 cctttaagct ggtcccagtt atattgggttactctcttct tagtctttgg aatttttctg 23700 atgaaaacct tttaaccttt atactgaacagggcattgtc taaatatagg agcagatctg 23760 cagatggggc caagaattac ttcgaacatgaggagttaac tgtgagccag ctcctgctgt 23820 gcctaaggga agggaatcag aaggtggagagacttgaagt tgcactcaag gaggccaaag 23880 aaaggtatga aataggttaa cttgaaatatgtgttttttt aaaacagctt tcctgagata 23940 taattaagat accatacagt tcacccatttaaagtataca tttcagtgtt ttttagaata 24000 ttccaggatt gtgcaaccac tgttactacaatataatttt agaacatttt ttcccccaaa 24060 cagcactcac tgtctgctcc tccaagcaatgtgctttctg tctctataga tttggccatt 24120 ctagacattt catataaatg gaattatacagtctgtggtt ttttgtgact ggcttctttc 24180 acgtagcata atgtttttga ggttcatctacaacgtagca tgtatcagta cttccttttc 24240 cttgctgaat aaccttccat tgtctatatatacaacattt tgtttattca ttcatcagtt 24300 gataaacatt agagttgttg ccactttttacctattagga ataatgctgc tatgaacagt 24360 gtgtacaagt ttttactggg atatgtgtttttaattctct ggggtatatc gttatgggtg 24420 gaattgctgg gtcatacggt atcgctatttcatattctaa ggaaccagca aatcattttc 24480 caaagcagct gcgccatttt gcattcccaccagcagtgca tgagcattcc actttctcca 24540 cgtccatacc aacacttgtt tcttactgttttcactttga cttcagccat cctagtggat 24600 gtgaattggt atctcatagt tctgatttgtatttccctag tgacagcgat gttgagcatc 24660 ttttcatgtt gaccgtttgt ttgatttggagaaaagtcta ttcagagcct ttgcccattt 24720 ttaaaaattg ggttatttgt ctttttatgttgaattataa gagttcattt tacattctgg 24780 atacaagacc cttattaaat atatgatctgcaactatttt ctttcttttt tttttttttt 24840 ttttgagacg gagtctcgtt ctgtcacccaggctggagtg cagtggcgca atctcggctc 24900 actgcaagct ccgcctcccg ggttcacgccattctcctgc ctcagtctcc cgagtagccg 24960 ggactacagg cgcccgccac cacgcccggctaattttttt tttttgtatt tttagtagag 25020 acggggtttc accatgttag ccaggatggtctcgatctcc tgacctcgtg atctgcccgc 25080 ctcagcctcc caaagcgctg ggattacaggcgtgagccac cgcgcccggc tgatctgcaa 25140 ctattttctc ccattctgtg gatcgtcttttcccttgatg gtatcatttg cagcacattt 25200 gtttttattt tgatgtaata cagtttatctcctttttctt ttgtcacctg tgcttttgat 25260 gtcccatctg agaaaccgtt gcctaacccaaggtcacaaa gatttactcc tatgtttttc 25320 tcctaagaat tttgtagttt tggcctggcgcggtggccaa aattacagtt ggccaccgca 25380 ctccagcctg ggtgacagag tgagactctgtctcaaaaaa aaaaaaaaaa gaattttgta 25440 gtttcatctc tgacatttaa gcctgtggtccattttgagt ttgtttctgt gtatgttgtg 25500 atataggagt tcaacttcat tagactctcagttctgtttc attgatctat gtttgtcctt 25560 acgccagtac cacaatgtct tgagtactatagctttgtag taagttttga aatcaggaag 25620 tgtattagcc cgttttcata cttatatgaagaaccgcctg agactgggta atttataaaa 25680 gaaagaggtt taactgactc acagttcagcatggctaggg aggccttggg aaacctaaca 25740 aatcctggcg gaaagcgaag gggaagcaaggcaccttctt cacaaggtgg cagaaaggag 25800 aaggaacgca ggaggcacta ccacacacttagaaaaccat cagatctcat gagaactcac 25860 tcactatcac gagaacagca tggaggaaactgcccccgtg attcaattac ctccacctgg 25920 tctctccctt gacacatggg gattatggggattacaattc aagatgagat ctgggtgggg 25980 acaacaaagc ctaaccatat gaggaaggagttaactgtga gccagctcct gctgtggcta 26040 agggaaggga atcagaaggt ggagagacttgaaattgcac tcgagagata gtgccttcca 26100 actttgttct tctttttaaa gactgttggctcttctgggt tctttgctgt tgcgtaagaa 26160 ttttaggatc agcttgttaa tttgtgaaaaaagccagctg ggatcttact agggattgca 26220 ttgtatcccc aaatgatttg gggagaattgctatctccag gattgtgtca tcgcagagat 26280 agtcttgctg cttcttttcc agtctgaatgccttttattt ctttttcttg cctgattatc 26340 ctgtaaaaaa aaaaaaaaaa aaaaaaaaaaagtgttacat agaaatggag agagcaggca 26400 tccttgtctt gttcctaatc ttagggggaaagctaaggcc ccacctttgt catcacttca 26460 gaggttcctg gtgtcactca tgcttgagtgtcctggagtt cccacagtgt gaatctggtt 26520 gcttcttggc tgtccctact gttggctcaaaggtcggcct tcttgggctg gtaaggcccc 26580 actcagacct gtactgccaa attttcctactattatttcc tctccctttt tctttgttcc 26640 tgtaggcaca tggctttttg aaggtccttttagtagagtt tgggctggga aaaaaattgc 26700 atgcatctgt tcaacccatt atctttaaccacagtctctt gtttcatttg gattgggacg 26760 gctttcctgt ggttatgatt tggtgttaagaatggtgtta ctttttttgt tgtcgtttat 26820 tcggtgactt ttaaacttag ctgtgtcctaaaaggaaaag tctttccttc tctaatgaat 26880 tcttatgaat gagataccat gttcatggaacacacatgca tccacatgtg taaacacaaa 26940 caatttcaaa aacattgctg cataggacagttgcatggaa acaaatggtg ttcaagatga 27000 gtttcacttg ccttttacct ctgtgtgtatttgtctgtga atcaattcta gccaatttta 27060 ggatgaaaaa taaaactaat gctaatatagtgaatgtgta gagattttga aaacccctga 27120 tcctttatcc caattgtaaa caatgttctttttagtactt ctgtaataat tgctatttct 27180 cttaaagcca aagagaaagt aacttttctatcttctgtga ttttccagag tttcagattt 27240 tgaaaagaaa acaagtaatc gttctgagattgaaacccag acagagggga gcacagagaa 27300 agagaatgat gaagagaaag gcccggagactgtgagtcct aagattccac ggccactacc 27360 acacccacac acacgagagt agtccagccactgaattcaa atcttgtgat gggttatttg 27420 ctttagaaat atagaaatca tgttgatattgaatattatc tatctattcc ttttatatgt 27480 ccttgtcctg ctctgtgtca attgtagcgagatgtatttc ttttttgttg ttgttgttgg 27540 agatggagtc tcactctgtc gccaggctggagtgcagtgg cacgatctca gctcactgca 27600 acctccgcct cccaggttca agcagttctcctgcctcagc ctcccaagta gctgggatta 27660 caggtgcccg tcaccacgcc tggctaatttttgtattttt aatacagaca gggtttcacc 27720 atgttggcca ggatggtctt gatctcttgacctcgtgatc ctcccacctc ggcctcccag 27780 agtgctggga ttacagatat gagccactgcgcccagctgc aagatgtatt tctatcagta 27840 ttctacaaaa cgatttccta tgtctcttcttgactgattt cttctcctcg gtccttcaat 27900 gaacaagcct actgtaggaa aaggaatgttgtccacttta taatgagatc atttgaggat 27960 atgacttaga aacttgaggg agaattgaaagatttgggtt ctatcccatt actggtttga 28020 ataaagtagt ttgaaaggaa aaggttcactgttgtcttgt tcagtgttgt ctggtaattg 28080 agagaggtgc cttcgagtct gcagagagtcttcagctttc ggaagttaat gaagccgtga 28140 aggttgatag ccataggggc ccacgtgaaaggcatttaca taaaatattc actttaggta 28200 attaatttat tcaacaaata tgtacattgaatgcctattg tatgtcagga gactgagacc 28260 ttactgttga aagaagagag aacatttagaaaacagatga agagaccacc agtgaataat 28320 agttccctgt tgactaaaac gaattcaacagccagtagca gggaaatatg gtctttcaag 28380 gcatcagaaa ctcatttaca aaaattatagagctgccagg aaaaaggctg cacaacaaaa 28440 atagttgagt aaactagaaa catacactgggaagagagta tgggggcaag ttgttagctg 28500 gatagatagg actgtgcttt gacacctctgtggtctatga tctctgaacc tggaataggg 28560 ttcattttaa tagcgataaa gtcattatcccagtgcatcc aaattgatta gttcatgctt 28620 tattaggaaa cagaagttac ccaaaacttagcaaacctaa gtaccaagta tccaaaacat 28680 tcttttccta cacaatgttt ggggtattgtcaaagttgga ttgattcacc agccagtctt 28740 aattggctac taatggttca gcctgttttctcctaaagag gtttgtttaa tgtcagatga 28800 taattgtaca gatatgtttg ggatttcccgtatgataggt tggaagcgaa gtggaagcac 28860 tgaacctcca ggtgacatct ctgtttaaggagcttcaaga ggctcataca aaactcagca 28920 aagctgagct aatgaagaag agacttcaagaaaagtaaga atgagagagc aattttatcc 28980 tcctttgaaa tatacatttt tacaaagtatactactatat aaaaacatag ttttttaact 29040 atgttatgac taaaagaaaa atagacacctaattaaaata taaattcaga atatactaat 29100 gttccagtta atgtgtgagc atgaaatacttgtaagatgg ggggttgggg actggagaac 29160 tttaattctg ccatttaggg gcatttgttaaatgtacgag cctgggtaag atctctacag 29220 taaagctgtg agctagtttt cctgttactgacttaagctg atgacattga tgtgagtaag 29280 cataaagaaa gatgaaaaga gcataaagatcttgagtgac atttatttgg aaaaaggtca 29340 atttcaattt gttatttcaa tcagttaattatttcaggct aacatgtaga ttgagcgttt 29400 ggcatttgct tgtttctctt gatgtaagaagttacccaaa acttagcaaa cctaagttga 29460 tgctgttcta ttggattcat tggcaaacatgtttctagca caatacggag gtgtgtgtgt 29520 tttcttgagg ttataattcc caacactctgtagaattatg gtaacgtgat acaacatggc 29580 agctacaact aaggactttg gacaaacagacctagattta acatatgagt ctgcaactta 29640 tttgtgttgg gcaaggtatt tatttcaattctctgagcct gtcttggcat ttgtaaattg 29700 tgtgttgtaa tttcttctat atacattgtgttaaatgata tgtctaatgg gctgggcatt 29760 aatgctttgt gcatagctgt catttatttgtattatattg aaatcctctt tccgatcttt 29820 aagaagactt aggggaactt cctttttcccttattgaatc tttgtcagaa actaaagtct 29880 ttgcaattga cagaacctat aactttttttttaatataaa agatatccac acatcactac 29940 atgagaagcg ccttagctaa ttactactgtggtctgtgtt taaatactaa aaatgtatct 30000 gtatgactag tttaaacaat tattcaaagaggacagtact gcatgtgagc ttagatctgt 30060 acttttttat gtttaggcgt aagggttcagaaatatggcc aggtctagtg aagaagcaag 30120 gaggattatg tatttcattt tgcattcataaaccctacag ccctaaaatt cttatattgt 30180 acataacctt ggggtttgtt taaaagccactgcgacgtaa aggagcattg tttatcctca 30240 tgaaatcttg acctttctta ggtgtcaggcccttgaaagg aaaaattctg caattccatc 30300 agagttgaat gaaaagcaag agcttgtttatactaacaaa aagttagagc tacaagtgga 30360 aagcatgcta tcagaaatca aaatggaacaggctaaaaca gaggatgaaa agtgagtatg 30420 ttgagtcaga agggcagcga cggggcagaggagggagaat cgccttttta tacagattgg 30480 aattcggatt tgagaataaa ttttaaaaaatttctttttc acttatctga aggagtccta 30540 gcagacctct cagagagggg gataaaatttaaaagttttg tcataataaa attatgctga 30600 ttgtttgcac tctgtcttga tttttcagaaaagatttttt ttgagagtaa gaaatgctag 30660 taggtcgtgg ggtgataaag gtaggcgagaagatttttct actggagtgt tcagaaggtt 30720 gggaggcaag actataagtt tctatgatattttccccagg attccatttt ttaatatctt 30780 ttttaatagg tccaaattaa ctgtgctacagatgacacac aacaagcttc ttcaagaaca 30840 taataatgca ttgaaaacaa ttgaggaactaacaagaaaa gaggtattca ctgaaaaaaa 30900 ttacttccat agcctagtaa tgaacagaaactgttgaacg ttttgtatat aaaatagtta 30960 catgaatcct tcactaaatc tggtttcaaaggttgttttc caatgtatca ttatttcttg 31020 catctagggt ttgtaacttc tgatgttccacatatgtgta atgtgcttta ttgcgtacaa 31080 agatgatgtg aatgtcctat ggtcagggattaagcacttc gtatttcttt tttttttttt 31140 ttgagacgga gtctcgctct gtcgcccaggctggagtgca gtggcgcgat ctcggctcac 31200 tgcaagctcc gcctcctggg ttcacgccattctcctgcct cagcctcccg agtagctggg 31260 actacaggcg cccgccaccg cgcccggctaattttttgta tttttagtag agacggggtt 31320 tcaccttgtt agccaggatg gtctcgatctcctgacctcg tgatccaccc gcctcggcct 31380 cccaaagtgc tgggattaca ggcgtgagccaccgcgcccg gccagcactt cgtatttcta 31440 aggataggtt tgtaggagag ctaagagcatgggcttctat gggtaggaag ggccatctgc 31500 tctggggaat tgtgcaagac cagcgtgcctgctgtcagtg aatttgggac cctggaatca 31560 tcagcctgca gtttaaattc ataataatggaccaggtgtg gtagctcatg catgtaatcc 31620 cagcactttg ggaggccaag gctggaggatcatttgaacc caggagtctg agaccagcct 31680 gggcaacagg gaggccctat ctctacaaaaaataaagagt taaccaagtg tgatgttcgt 31740 gcctgcggtc ccagctcctt gggaggctgaggcaggagga tcacttgagc ctaggaggtc 31800 aaggctgcag tgagctgtga tcacgccactgtactccagc ctgtgacaga gtgagaacct 31860 gtctctaaaa aaagaaaaca ataaataaattagtaataat gccagcatgg tgtgatagtt 31920 tagagaccac agaagcttgt gaattagaagggatctttga atttttagcc ttgtaaatat 31980 actctttgtt tttcatttat tttcttttaaagaggaggtt ttgccatgtt gcccagaatg 32040 gttttgaact cctgagctta ggcaatccacgtgcctcagc ctcccaaagt gctgggatta 32100 caggtgtgag ccatcatgcc cagcagtagtgttcctctct tggacctaat aattttaaat 32160 ttaaaacatg tttcttcttt tccactgactgcaggaagta acaagtggca aaataacagt 32220 atcaacgagt cacagcctta ttaacattggagtttgttat tgtatccctg atttcggtgt 32280 tatcaccttt tttttaggaa ttcattatttgcaagccaca acttaaatac aactttctga 32340 ataagttagc gttgctgatt aatagactggttagagctga tacatttttt agatctcgct 32400 atgttgccca ggcttgtctc ccactcctgggctcaaacga tcctcccacc tcagcctctc 32460 aattctaggc atgagccacc acacccggccagagctgata attaaaaaaa taaacctttt 32520 tctaatattt tactaaaaca ggcagaattatttcaaaacc atttctagaa taaatgtttc 32580 tttttcagtc agaaaaagtg gacagggcagtgctgaagga actgagtgaa aaactggaac 32640 tggcagagaa ggctctggct tccaaacagctgcaaatgga tgaaatgaag caaaccattg 32700 ccaagcagga agaggacctg gaaaccatgaccatcctcag ggctcaggtg aggcaccttc 32760 caaaacccca gctgagcgag gccagccctgactgtattct cgcattggaa agcaatggtg 32820 tttagaatgt ttgtaatttt ctattttatatattttttca cccgtgagtg tattaaaact 32880 ttaaaattga aacatttgga aagtgctcagtggatcttat ctgttctaca tttaataggt 32940 aattggattc tttccagttt gtggcattatgattaacgtt gctaagacat tcctgtgcat 33000 gttgctctgt tcacatgtgg atattttatatttctgttgg gtacacacct aggagtggag 33060 tcgctggatc ataggctctg catgttactcacttttaaca ggtaatgcca aacagttttc 33120 cagagtggtt ggaccagttt tcactcccatcaacagagag tttccatggc tctacatctt 33180 accaacactt ctattatcag tcattttcctttaaccactc tggagggtat atagtggtat 33240 ctcatttaat ttgcatttcc ctgatcactaatgggaaaga gtactttttc aagtgttttt 33300 ggcctttgag gtatcctctt ttgtgaagtgccttatcaag cctgcctttt tttttttttt 33360 tttttttttt ttttttttgg tttggtttgcctggcatttt ttttgagaca gagtcttgct 33420 ctgtcgccca ggctggagta cagtggtatgatctcggctc attgcaacct ctgcctcctg 33480 ggttcaggtg attctcctgc ctcagcctcccatgtagctg ggactacagg catgtgccaa 33540 catgcctggc taatttttgt atttttagtagagatggggt ttcatcatgt tggccaggct 33600 ggtctcaaac tcctgacctc aggtgatccacccacttcca cctcccagag tgctgggatt 33660 acaggcatga gccactgcgc ctggcctggctttttaaaaa aatgtaatga cttctatgta 33720 tactgcacat acaagtcttt gtcagttatttttgcctttt cactctcata aaggtgtcat 33780 ttgaggaaca aaagttctta gttttaaggtagtccagtat atcagccttc tcatttatga 33840 ttagtacttt ttgtgtcctg tttaagaaacttttactacc ccaaggccag gaagatattc 33900 cctctgttgt cttctaaaaa ttttgttgtcttacctttta cattaagatg tacaaaccat 33960 ttgaaattgg cttttgtcta tagaaaatgaggtaagggta aatatttatt tttttcctat 34020 atgaatatcc agttaacttg gcaccatttaagccatcttt tcttaagtgc tctgctgccc 34080 tcttcatcat aacttgtgtc catatatgcatgggtctgtt tctggatttt gttctatttt 34140 attggtctac tcatatatct ttgctctggtaccatgctgt cttattataa agcatcaaac 34200 tttttttttt tttttgagac agagtctagctctgtcacca ggctggagtg cagtggcacg 34260 atcttggctc actgcaacct ccacctcccgggttcaagcc attctcctgc ctcagcctcc 34320 caagtagctg ggactacagg cgcacaccaccacgctcagc taatttttgt atttttagta 34380 gagatggagt ttcaccacgt tagccatggtggtctcgaac agttgacctc atgatctgcc 34440 cagctgggat tacgggcgtg agccaccgtgcctggccagc atccatttga atggttggat 34500 catgagttat gtccatttct cagttaaataaatattacca agctgtcttc taaaactgcc 34560 taaccaaaat tatactctca ccaaaagataagcactccta tgtcctcaca ttcttgcatt 34620 attttaaaaa ccttggccaa tctgatgaacatattatttt taatataata atgtcagcag 34680 tttgttattc cctacataca gactcattatcaccagctgc ccagagtggc tgacccctgt 34740 gaaatgcagc cagttgactg gtacttatagctttagtttt atttctacag gaatagagcc 34800 tcaacctgca ttctcagctc ttcagagcatttccttgctt gggtaactca tgtcattggg 34860 ttcttccact ctgctgcagt catgtatttttgctcattct agacatagac atttgttttc 34920 tcttgatgga tactcttgct cattcattcaataacttctt ttttgagcac ttataatatg 34980 tcagagacgg ccgggcatgg tggctcacacctgtaatccc agcactttgg gaggccgagg 35040 caggcagatt acaaggtcag gagttcgagaccaacctggc caatatggtg aaaccccgtc 35100 tctactaaaa atacaaaaat tagccgagcgtgttggcggg cacctgtagt cccagctact 35160 cgggaggctg aggcaggaga attgcttgaacctgggaggc agaggttgca gtgagccgag 35220 atcatgccac tgcactccag cctgggcgacagagcgagac tctatctcaa aaaaaaaaaa 35280 aaaaaaaaaa aaaaaaatca gacactgttcttactgcctg ggatatagga atagacaaaa 35340 caaagactct gttcttactg gctcaaagctagtaagtgat agaagtgaga ctaaatcgaa 35400 tttaagtcat actccaaaac acccatgatccactgccaac actgtgagat gctgaaccac 35460 tcacattgat aggatatccc ttaaaatgatctctttatta catactttta agtaactata 35520 gttttgatag aatatttaga tttttaattttacttttaaa ataatggata ataatttata 35580 tctcagaacc agcaaatttg aaaagaaaaaactaatttta aacatagaaa gtgaaaaatt 35640 agtattaaat tccaatacca atggaacagttcactcaatt agctgacagc attaaatatt 35700 aaaacactat taagtgtttt gttatttataattttttaaa aattacttaa tttttaaaat 35760 gttgtgttgt tttgtttttt gaggcagggtcacactcctg tcacccaggc tggagtgcag 35820 tcgcatgatc gtggttcact gcaacctctatttcctgggc tcaggtgatc ctcgtgcctc 35880 agcctcccaa gtagctggaa ctacaagtgcatgccatcat gcccagctga ttttttgtat 35940 ttttagtagc gatgaggttt tgccatgttgcccaggctgt cagcaggagg cctcagttcc 36000 tcaccatgtg gtctcctcca gagggatgcttgggtgccct catgtcatgg cagctggctt 36060 cccccaaagc aagtgatcca acagagagcaaggagtgaca gcgtcttttg tgacctagtc 36120 tcagaagccg tacaccatca cttctgcggcatcctggagg ttacacaggt tgggtttatt 36180 cattgtggga gggaaacata caaggtgatgaataccaaga ggcaaggatc actggagcca 36240 tcttaggagg ctggtgacca cacatgggacatggattgaa tatggaaaag ttcatgggaa 36300 atggtcttct gtctggttca aatgctgatttggccactta acaaattttc ccaagattaa 36360 gagccgttaa gtttgtaaaa tgaggatagccattctttat tctcaggata tttcataagg 36420 taaaataaga tagtaaatgt aaagcacccaacataggacc tcacacatgt ttggaattta 36480 acaaatagca tctatttgtg atgattattcttttaaattt agcttaagac cagccttcat 36540 aaatacacct ggcagaatca atttactatattaagtaatc atttactata ttaagttgat 36600 cctgaattgt ttattatcta aaagtccagataattttgct gaattaatgg tacctacagt 36660 atttaaacta cctatatcag tgcagttgcaggatttgtgt tgtttaaagc acacacacaa 36720 acacagcttg tatctgctat cggaatgtacctggaaagtc atggtcatta tactgttttc 36780 tagcaggatt gtgcatctgt gattcacaagggctattgaa ggatacagca ctacctcctc 36840 atcgcataaa cactgtaaga atctgcattcatctaggtac taacttctgt atcttttttt 36900 cctctaacag atggaagttt actgttctgattttcatgct gaaagagcag cgagagagaa 36960 aattcatgag gaaaaggagc aactggcattgcagctggca gttctgctga aagagaatga 37020 tgctttcgaa gacggaggca ggtaaggaaaagagagagga ggacccagag ctcacatcag 37080 catggccgta gaagaggtgc ctgtccaaagacgttcctga tttgaactat aagaatagct 37140 gtgttcgcgc cactgcactc ctgcctaggtgacagagcga gtcccctgtc tgaaaaataa 37200 ataataataa taataattgc ttcacttacacttcatgtga tcatgttccc aacacttagt 37260 ttgtcttaca ggaaagcttg acagagacttgtgggagctt gatcaagctc cttgctttta 37320 gataagcaag gattttgatt tgattttaaaatgttgtgtt gttttgtttt gttttttgag 37380 gcagggtctc actcctgtca cccaggctggagtgcagtgg catgatcatg gttcactgca 37440 gcctcaactt cctgagctca ggtgatcctcgtgcctcagc ctcccgagta gctggaacta 37500 caagtgcatg ccaccatgca cttgtaacaataatgttacg tgtcccaatg acctatcttg 37560 ccatcgtcac ggatgaaata ttcgtagcactttaaattcc tgaatattct ttaaaaaata 37620 ctaacaaaaa ttacttctga cttttagaaatttattttga gaaagtttta aaacacagca 37680 aaattcagag aataaaataa cagactctactatgtactcc ttcctgcatt gacaactgct 37740 tttatttgct tcaagtattt tttttttattaaaggaaaaa gggagttgta gttagaatca 37800 aagtctcctt tgttccccat ccatcattatattcctttct tctttacctt gtgctgttag 37860 gaatttggtg ggtagcttcc ccatctattttatactttta catatcacat acacacttac 37920 ctatatcata tctcaaaacc agataatattgatttctctg tgtttaagtt acaaaatgat 37980 cactgtaggt attgttctgc agcttactttacataatatt atgattttga gctctcttga 38040 tatgtgcgga tgtaatttat tatacttcattgctgtattt tgatttataa atatgccact 38100 tctttctaat ctgtttccta ctgatgacagtttggttatt tcctgatttt ttttaactgt 38160 aattatttac tttcactagt ctcctagtgccaatagtatt taaaactaaa attagtctgg 38220 tttttatgaa ccttggcagt gtagtttgagtcttttttcc cctacttctg tggactgtct 38280 gctcagtgtt gtcatgtttc ggggttgtagaacatcacac agcgtgttgc ttttcgtcct 38340 ggcaggcagt ccttgatgga gatgcagagtcgtcatgggg cgagaacaag tgactctgac 38400 cagcaggctt accttgttca aagaggtgagtcccgtgtga tcctggattt tcaggaaata 38460 gctatcctat gaaaaagatg cttgaagaaaaattccactt cattctctac aatggattcc 38520 aaatcaaggc accaaaaata tagcacccgtcagtctcatt accacagcac tcccatctcc 38580 atccattacc caccgaatcc agaccagacccttcaccctg ccagaaggtg cctggcacgg 38640 ccacactttt tctttttttt ctttttttttgagacagaat ttcgctgtgt cgtccaggct 38700 ggagtgcagt ggcgagatct cggctcactgcaacctccac ttcctgtgtt caaacggttc 38760 tccttccaca gcctcccgag tggctggaattacaggcgtg caccgccaca cccagctaat 38820 ttttgtattt ttaatagaga tggggtttcaccgtgttggc caggctggtc tcgaactcct 38880 gacctcaact aacctgcctg tctcggtctcccaaagtacc gggattacag gcgcaagcca 38940 ctgtacccgg cctacagcca cacttttaaaccgtgtctcc ctctgttctc ttacagacat 39000 tagccagact gaatcatccc cttgaacttgtcctaagctt gtatttgctt gtattatgcc 39060 cttcacagag acgccctttt cttatgcatattcctgtctt cctccagtct ctttccagtg 39120 acctcttacc ccatctttaa aatctatttcaaactccatc tccatgaact gtttccattt 39180 tgaacttcca aagtacttta ttcctctatgttggtacctg tctgctattt tataggtgtt 39240 tgtgtgttct aggtagatac ttaagtactttatttttatt tctattttta ttttgagaca 39300 gggtcccact ctcttgccca ggctggagtgcagtggcatg atcgtggatc actgcaacct 39360 ccgcctcccg ggttcaagcg attctcctggctcagcctcc caagtagcta ggactacagg 39420 tgcacgccac cacgcctggc taatttattttagtagagac agggtttcac catgttggcc 39480 aggctggtct caaattcctg accccaggagatctgcccac gtcggcctcc caaagtgctg 39540 ggattacagg cgtgagccac cacgcccggctaatttgtat tttcagtaga gacagcgttt 39600 caccatgttg gccaggctgg tctcaaactcctgacctcag gtgacccacc cacccaggag 39660 aggtctccta gaacagaatg taccttctcagaagcagagg caatggtctc atttattcag 39720 cagatacttt tgagtcctcc aggatgtgcaagaggtactg cttatgctgg gagtcagaga 39780 gcaaaggcag ccccggatgc tgacccctcttctctggcaa tggggtgatt agaaagctcc 39840 ctgctttgga aaccatgctt tatgatcaatacatacctct tcagaaatgc ttcttaaggc 39900 tgctgaacac aatctcaaat attcatacatgttactacat ccaagtaatg tacttagaag 39960 caaataaagg ttgcctccaa aaacaaacattttgagagcc aaaccaaagt ctaggctgag 40020 tccaaaaaac caagacggtt tgcattggtccagaaaggtc agaacagaga gatgcccggg 40080 agccagaaat aatttcttgc atccatcacagtttgtatgg atccaggacc tagctagggt 40140 cagggcttat ttcgaacatg ctctgattgaggcttgaacc tcagtactac tccaaaactg 40200 ggggaaatga tagggtgcat tcttccatcattcctccttg ttaactttat gtagataact 40260 cctgttcatt ctcaaggccc ctctcaagttaattggtcgc atctaggagg cctttctgca 40320 ccaagatcta aactaggggc ccttccctttctcccatgga cacccttgca cctaacactc 40380 acctggccta acactcgtgc ctaacacttttgccacgcca ctgctgccac atctctcacc 40440 tggcttctcc accacgtctg tctcccggcacacagcagtg tctttataca tcccagagga 40500 ttcaaaagtg tctgctgaac aaatgagatgattgcctctg cttctgggaa ggtacactct 40560 gttctaggag acctctcctg ggctctgtggatttctttct ccacattccc ttcctctttt 40620 gcttatatcc tcattcatcc cttttcttctcgcagatatc ctagtattta ctagaacatg 40680 cattgtcctg tacctagaac tgaaaccatgtattttcgca taccttgaaa ctgggataca 40740 gtttaaatcc taaaagaatg caccaatgcacctgtaatcc cagtacatgg gaggctgaga 40800 tggaaggatc agttgagacc aggagttcagaaccagcctg ggcaacatag tgagatccca 40860 tccctacaaa aattttaaaa actagccaggtgtggtggca cgtgcctgta gtcgcagcta 40920 ttcaggaggc cgaggcagga gggtcactggatcccaggag ttagaggctg cagtgagcta 40980 tgattgtgcc actgcactct agcctgggagaaagagcgag actccatctg tcaagaaaaa 41040 aaagtaaaga aaagaaaaaa atcctaaaacaggccaggcg cagtggctca tgccagtaat 41100 cctagcactt tgggaggcca aggtgggcagatcatgaggt caggagttcg agaccagtct 41160 ggccaacatg gcaaaaccac atctctactaaaaatacaaa aattagctgg gcgtggtggc 41220 gcgcacctgt gatcccagct actcaggaggccaaagcagg aggatcactt gaacctggga 41280 ggcggaggtt gcagtgagcc aagatcgtgccactgccctc cagcctgggt gacagcgaga 41340 ctccgtctca aaaaaaaaaa aaaaaaaaaaaaatcctaaa ataataggga agcaggtatc 41400 acttggagag atttttctct atgtgcatcgtgatgacttc agttaaagac caaacacctg 41460 tgctcatgtc ccactacgtg ttgaatacgaagttgaactg atgttaaaac tcgccatctg 41520 ttcttcaagt gaaacaaaca caactgcctgcaaaatggaa ctaatggaat tatcatactt 41580 attcccagga gctgaggaca gggactggcggcaacagcgg aatattccga ttcattcctg 41640 ccccaagtgt ggagaggttc tgcctgacatagacacgtta cagattcacg tgatggattg 41700 catcatttaa gtgttgatgt atcacctccccaaaactgtt ggtaaatgtc agattttttc 41760 ctccaagagt tgtgcttttg tgttatttgttttcactcaa atattttgcc tcattattct 41820 tgttttaaaa gaaagaaaac aggccgggcacagtggctca tgcctgtaat cccagcactt 41880 tgggaggtcg aggtgggtgg atcacttggggtcagggttt gagaccagcc tggccaacat 41940 ggcggaaccc tgtctctacc aaaattacaaaaattagccg agcatggtgg cgcatgcctg 42000 tagtcgcagc tactcgcgag gttgaggcaggagaattgct tgaacccagg aagtggcagt 42060 tgcagtgagc cgagacgaca ccactgcactccagcctggg tgacagaggg agactctgtc 42120 tcgaaagaaa gaaagaaaaa aaggaaggaaggagaaggaa ggaaggagaa gaaaaggtac 42180 ctgttctacg tagaacacct ttggtggagttccatcaact cgcaaagtag aatccttacc 42240 tactactctt ctgataataa ttttaatattttttatgttt ggttgatgcg agcagctgca 42300 ctgctcatgc agttagctag catgtgacatcatgtgacaa agttcatgta attagatgga 42360 agaaacctca ctgattaatt ttaagaaccttttagggatg caggaacaat gaagtggcca 42420 cagtatgtgc tgtttttgaa gcatttttaaaaacgaattg tagttgtttt tcttcattta 42480 aaatggatct gttggaggtt atgtgtgtatgttgtagttt tattgcagcc acaataattt 42540 taccaaagtt ttcacatagg cagttagcctttacttaata tcaagacaag tgaaaaaata 42600 ttggcatcga tgaaaccgat aacattggcctcattggatt tctttaccca ttcacagtgt 42660 aaagaagtta ccttcatgct ttcattgtacctgcaggcct gtgggcttgt acagtagata 42720 attaatttct aaaaagaaca gctgcctattttcttcctag gttaggttat atcttcataa 42780 tcacaagaat tagtgatggc aaaataaaattttgcttatg aatcttttac attgtttata 42840 tatgattaat atcatcatat atattttctgtattaagctc atttggcttc atttaagctg 42900 tatacttagt catatatctt tcattagttctatggatatg agcagatccc tttactggag 42960 cccagtatgt gctgtgtgag ttagaagtcattcttgctga gaaggtgaat aggtagggat 43020 ttgccttgtt ttgtaagtct acaatttgccaagagtaaat aacactggac cagctgtaaa 43080 agtaaacagt gtgtttatgc attgagatactaaagcattt aagaaaaaat taaaagatct 43140 cttttgttta aatttgtctt aagaatctctcctagacaaa cttgactata cacacacaca 43200 cttcaataag aatcaaaata gttcattatatcttcatgcc aagagaacac atgatacaaa 43260 ttcaacagta atttcaatta aacgagtcccctaccacacc tcagggagaa tatttgttaa 43320 agacatttct aaagtaactc tgacatttttacttttcttt gttatggttt ctaatgtaaa 43380 ttttttccct tatcatctta tactggaggaaatatatttc tatagtctcc taaacagaga 43440 aggaaaccaa atggtttcac atttaaagttattggacata atccttttac tttgttcaag 43500 taaaaatatt cagcaaagcc agactgaaataataacatga atgggcttat taagcttgtg 43560 accaaatcgt ttgtttaata gttggaagaattacaaatga aggccatttc acgtagtcag 43620 tttaaattaa ggctgttgtt tccagcttggtaaatagctt ataaactctg tgcacatctt 43680 acctcattta tttattctaa tatcccttcaaaggctctgt tccatgtgac cagttaacta 43740 tacctcctag gaattttgtg tctgtaaaaatacatagttg aagattgcac tcttaacagg 43800 tttacgacta tcctggatag tattgttaactgtgtgcaca ttgttgtaga gcagatctct 43860 agaacttttt catcctgcgt gaatgaaactctacacccat tcaactgcag cccccattct 43920 cccctcccca gtccccggca accaccactctcctttctgc ttctgagttt cacatacctc 43980 ctataagtgg gattacatag tatttttccttctgtaactg gcttatttca tttaacatac 44040 ctccctcatt caaaccataa cttcctggatgaaggtttca ggcaaaagca cctgggaaac 44100 caggcctttg ctactcattt accccccattccccacccca ccccccgccc cagtcccgtg 44160 cctccacctg ctgagcaccg ttccatttgccgatgctctc atgttaccct gggagacata 44220 aggtagaaga gccatgggga ctctgctcgagggtggtata aggcatttga aactaagcct 44280 cttcattaac ccaatccata tcacagagaggcagcatggg tgcccctagc ccaatggccg 44340 ggctatttgc tactgcaata ataggtttcttataaaccgt cagcctcctg agtagctggc 44400 accacaggga caccacaggc tattttttaaaaaaatttat gtagaaatgg attctcactg 44460 tgtttctcag gctagtctca aatttctaggctcaagcagt cctcccgcct cagcctccca 44520 aagtgctagg attattggtg tgagaggaatccctttcaac ccttgaaaat aaatcaagcg 44580 gtgatgaact ccctgagctt atatttgcctaggaaggtct ttatttctct ttcatttttg 44640 aaggacagtt tggctggaca tagtatgtcttatttggcag tttgtcccct tctgcactgt 44700 gaatatatta tgtcagtccc ttctggccatctggcaaggt ttctgctatg aaatccactg 44760 ttttatgaag gatctcttgt tcatgacaagttgctttttg attttaaaat gactcaatgt 44820 ggatgtcttt gggttcattc tagttggagtcctttggaat tctcgaatct ggatgtctat 44880 tttctacccc agatttggga aattttgagccattattttt tttaaagaag ctttctgtcc 44940 ctttttgtat ttcttcttaa attcccatagtgtttatatt ggtccactta atggtgtccc 45000 agaagttcct tagactttca tcattttaaaattattttcc ctttttgatc cactgactgg 45060 ataatttcaa ataacctctg agttcattctttcttctgct ttatcaaatc tgctgttgaa 45120 cccctctaat aaatttttca atttattttattcttcactg ccaacatttc tgtttggttc 45180 ttttttatac tctctctgtt gatattctcattttgttcat acatcgtttt cctgagctca 45240 ctgaacatct tttgaatttt ttttcaggtaagtcatatac ctctagttct tggggtcagt 45300 ctctagagat ttcatttgcc tctttgggccatattttcat atgtttcttc atgtgtcttg 45360 tgattttgtg tcgggatcca tgtatttgaaaaacaactct gtcttccagt ctttaaggac 45420 tggcttcata cagggaaaga tcttttccaatcatcttggc tagagattct gagatctccc 45480 agacctcttc catagataca tccccccagacctgtgcatg caaatgttca attagatttg 45540 ctagtctcgt ttttcacaat ctgcagcctcttgttccttc cagtgtctgc ctgcagccct 45600 gcatattccc tggagctgcc acaagccatccagcactcct tatcttattc tcagcagcct 45660 ctaggtctct ctctagagca ttctgggttctgtcaaaact ctttaagttg ggcgagatag 45720 aaccagttcc ctgggcagcc ccttaaaagccagaacattt gaaacacact ccactagtct 45780 ctttccctcc acaagggaga ggctggctgagctgtattaa cctctgtatg ctgcaccatg 45840 agtcctggag cagtagcagg tcaggtcacccagctctctc tctttcccag tgtcctccag 45900 gcatctagag tatgctgtgt tccatcagcactccaagaca aagcagaaac gagtccctca 45960 gacagcctcc caaaagggca gaatgttggacacactttgc tcttctcttt tcccctgtat 46020 aggagaggcc gccaagatgt attggcctctgttggactgc agatcctcta cagcagcagc 46080 aagctgccat gcttttgttc tcagtagtccccaggcatct atggtatatc aagcctttca 46140 atgctcaaga caagagaaac agtcccctgggaagccccct gaaaaactgg aaattggatt 46200 caggctccaa ctctctccct ccccagggaaaggcaagagc caggtgtttt ctcccacttg 46260 tttcacactg agctggggca atgggtatggtgacagagta catgccaatc ccagtctcct 46320 cttttgttct gagtggtccc tagactgatacccttttcag tcagttccta aattcaggca 46380 agagagaaac taatccctca ggcagccccctgaaaagtct gcacattggg cataggtatt 46440 agtcttctct ttgcctcccc agggagaggtcaggagctgg gagctttctc ctgattgtgc 46500 cacactgagc cacaggaggt actatggcaagggtatgcca caggttctcc tacatggctg 46560 gtttcatgcc catctggagt ggaggagcctgttaactatt ttcttgactt ctcacaaagg 46620 gaattcatcc atgtattgtt gaaccagtgccttcccggcg gggaaggagg gcctggggct 46680 tcctgttctg ccatctccct tttgtaaatgctgccatgct gtatactttt ttttttttaa 46740 aggataaaca tttctgttta aaggataaacattcaacgtt aactggaaat gaaaaggaga 46800 cgatttttag tctgatactt ataatgcaatattatttgca attctgtata aatagatttc 46860 agaaacttcg atttcaaatc ccaattaaattaaagagagg aaaattactg agaggaactg 46920 cagttccaaa tttttccttt cagggcagtca 46951 3 15 DNA Homo sapiens Putative OCTB/TST1.01 motif 3 cagcaattccacttc 15 4 22 DNA Homo sapiens Putative AP1F/TCF11MAFG.01 motif 4atgatatgac ccagcaattc ca 22 5 11 DNA Homo sapiens Putative GATA/GATA.01motif 5 tgatatgacc c 11 6 11 DNA Homo sapiens Putative EVI1/EVI1.05motif 6 agttatgata t 11 7 16 DNA Homo sapiens Putative FKHD/FREAC2.01motif 7 gaaagttaaa cagaga 16 8 13 DNA Homo sapiens Putative IRFF/IRF1.01motif 8 ggaaagttaa aca 13 9 11 DNA Homo sapiens Putative MYT1/MYT1.02motif 9 ggaaagttaa a 11 10 18 DNA Homo sapiens Putative XBBF/M1F1.01motif 10 gagttccttg gaaagtta 18 11 12 DNA Homo sapiens PutativeNFAT/NFAT.01 motif 11 ccttggaaag tt 12 12 13 DNA Homo sapiens PutativeIKRS/IK3.01 motif 12 tcctcggaat att 13 13 15 DNA Homo sapiens PutativeOCTP/OCT1P.01 motif 13 ccaaatattc cgagg 15 14 12 DNA Homo sapiensPutative PCAT/CAAT.01 motif 14 tggaaccagt ga 12 15 9 DNA Homo sapiensPutative AP1F/AP1.01 motif 15 ttgattcag 9 16 15 DNA Homo sapiensPutative BARB/BARBIE.01 motif 16 aactaaagct gagac 15 17 20 DNA Homosapiens Putative PERO/PPARA.01 motif 17 taaagctgag acaaagtcca 20 18 11DNA Homo sapiens Putative AP1F/NFE2.01 motif 18 ttgtctcagc t 11 19 14DNA Homo sapiens Putative HNF4/HNF4.01 motif 19 gagacaaagt ccag 14 20 8DNA Homo sapiens Putative SMAD/SMAD3.01 motif 20 gtctggac 8 21 13 DNAHomo sapiens Putative RORA/RORA1.01 motif 21 agaccaaggt caa 13 22 9 DNAHomo sapiens Putative SF1F/SF1.01 motif 22 ccaaggtca 9 23 16 DNA Homosapiens Putative AP4R/TAL1ALPHAE47.01 motif 23 tagggcagat gattca 16 2418 DNA Homo sapiens Putative AP1F/AP1.01 motif 24 atgaatcata tgaatcat 1825 10 DNA Homo sapiens Putative PIT1/PIT1.01 motif 25 attcatgcag 10 2621 DNA Homo sapiens Putative MINI/MUSCLE_INI.03 motif 26 tgcagcgaccacaccagtgg c 21 27 6 DNA Homo sapiens Putative HAML/AML1.01 motif 27tgtggt 6 28 16 DNA Homo sapiens Putative OAZF/ROAZ.01 motif 28ctgcagcaaa gggtgt 16 29 8 DNA Homo sapiens Putative MZF1/MZF1.01 motif29 gttgggga 8 30 15 DNA Homo sapiens Putative ETSF/ETS1.01 motif 30ccaggaactg gtttc 15 31 10 DNA Homo sapiens Putative RPOA/DTYPEPA.01motif 31 tccatgaaac 10 32 9 DNA Homo sapiens Putative STAT/STAT.01 motif32 ttcatggaa 9 33 12 DNA Homo sapiens Putative MYT1/MYT1.01 motif 33aaaaattgtc tt 12 34 12 DNA Homo sapiens Putative NFAT/NFAT.01 motif 34ccatggaaaa at 12 35 15 DNA Homo sapiens Putative SRFF/SRF.03 motif 35accatccatg gaaaa 15 36 10 DNA Homo sapiens Putative CLOX/CDPCR3HD.01motif 36 catggatggt 10 37 21 DNA Homo sapiens PutativeMINI/MUSCLE_INI.03 motif 37 ccaccccccc acccaccacc a 21 38 14 DNA Homosapiens Putative RREB/RREB1.01 motif 38 ccccacccac cacc 14 39 13 DNAHomo sapiens Putative SP1F/SP1.01 motif 39 ggtgggtggg ggg 13 40 13 DNAHomo sapiens Putative EGRF/WT1.01 motif 40 gggtgggggg gtg 13 41 14 DNAHomo sapiens Putative RREB/RREB1.01 motif 41 tcccaaaacc accc 14 42 19DNA Homo sapiens Putative SEF1/SEF1.01 motif 42 tgcctgatga tctgaggtg 1943 21 DNA Homo sapiens Putative PAX6/PAX6.01 motif 43 gatcatcaggcattagagtc t 21 44 19 DNA Homo sapiens Putative PDX1/PDX1.01 motif 44atgagactct aatgcctga 19 45 16 DNA Homo sapiens Putative AHRR/AHRARNT.01motif 45 tctaggttgc gtgctt 16 46 14 DNA Homo sapiens PutativeFKHD/XFD3.01 motif 46 attgtcaaca gaac 14 47 14 DNA Homo sapiens PutativeSORY/SOX9.01 motif 47 tgttgacaat aggg 14 48 15 DNA Homo sapiens PutativeCREB/TAXCREB.01 motif 48 tagggttcac gctcc 15 49 21 DNA Homo sapiensPutative PAX6/PAX6.01 motif 49 agggttcacg ctcctatgaa a 21 50 13 DNA Homosapiens Putative E2FF/E2F.03 motif 50 gagcgtgaac cct 13 51 16 DNA Homosapiens Putative AHRR/AHRARNT.01 motif 51 tcataggagc gtgaac 16 52 14 DNAHomo sapiens Putative OCT1/OCT1.05 motif 52 ctgcattaga tttt 14 53 18 DNAHomo sapiens Putative AP4R/AP4.03 motif 53 taatgcagct gctgatct 18 54 12DNA Homo sapiens Putative MYOD/MYF5.01 motif 54 atgcagctgc tg 12 55 14DNA Homo sapiens Putative SP1F/GC.01 motif 55 aagaggcgga gctt 14 56 13DNA Homo sapiens Putative EGRF/WT1.01 motif 56 gggtgggtga gca 13 57 9DNA Homo sapiens Putative VMYB/VMYB.02 motif 57 agcaacggg 9 58 20 DNAHomo sapiens Putative PERO/PPARA.01 motif 58 tcctgagagg ccacaggcca 20 5914 DNA Homo sapiens Putative HNF4/HNF4.01 motif 59 aggccacagg ccag 14 6016 DNA Homo sapiens Putative B2TF/E2.01 motif 60 aaaccccggg tggtga 16 6114 DNA Homo sapiens Putative RREB/RREB1.01 motif 61 ccccaaaccc cggg 1462 14 DNA Homo sapiens Putative GKLF/GKLF.01 motif 62 caataaagca gggg 1463 12 DNA Homo sapiens Putative CLOX/CDP.01 motif 63 ccaataaagc ag 12 648 DNA Homo sapiens Putative RPOA/LPOLYA.01 motif 64 caataaag 8 65 30 DNAHomo sapiens Putative HOXF/HOX1-3.01 motif 65 tttattggac ataattattaggtcgtgttc 30 66 11 DNA Homo sapiens Putative ECAT/NFY.02 motif 66tgtccaataa a 11 67 12 DNA Homo sapiens Putative PCAT/CAAT.01 motif 67tatgtccaat aa 12 68 16 DNA Homo sapiens Putative HMYO/S8.01 motif 68tggacataat tattag 16 69 8 DNA Homo sapiens Putative NKHX/NKX25.02 motif69 cataatta 8 70 26 DNA Homo sapiens Putative GREF/PRE.01 motif 70atattattag gtcgtgttct ttttgg 26 71 16 DNA Homo sapiens PutativeMEF2/MEF2.01 motif 71 caccaaaaag aacacg 16 72 8 DNA Homo sapiensPutative EBOX/USF.02 motif 72 ccacatgc 8 73 19 DNA Homo sapiens PutativeCDXF/CDX2.01 motif 73 ggtgaatttt atggcatgt 19 74 18 DNA Homo sapiensPutative MEF2/AMEF2.01 motif 74 tgccataaaa ttcacccc 18 75 10 DNA Homosapiens Putative RPOA/DTYPEPA.01 motif 75 gccataaaat 10 76 10 DNA Homosapiens Putative TBPF/TATA.02 motif 76 gccataaaat 10 77 11 DNA Homosapiens Putative EBOX/SREBP1.02 motif 77 attcacccca t 11 78 10 DNA Homosapiens Putative PIT1/PIT1.01 motif 78 aatcatacat 10 79 9 DNA Homosapiens Putative AP1F/AP1.01 motif 79 atgaatcat 9 80 16 DNA Homo sapiensPutative HMYO/S8.01 motif 80 ggctttcaat tacact 16 81 15 DNA Homo sapiensPutative OCTB/TST1.01 motif 81 tttcaattac actta 15 82 13 DNA Homosapiens Putative NKXH/NKX31.01 motif 82 ttttaagtgt aat 13 83 10 DNA Homosapiens Putative TBPF/ATATA.01 motif 83 ctttttaagt 10 84 11 DNA Homosapiens Putative MYT1/MYT1.01 motif 84 aaaaagttgt a 11 85 19 DNA Homosapiens Putative CDXF/CDX2.01 motif 85 tgatggtttt acaactttt 19 86 30 DNAHomo sapiens Putative HOXF/HOX1-3.01 motif 86 ttgtaaaacc atcattacaattcaaattta 30 87 19 DNA Homo sapiens Putative PDX1/PDX1.01 motif 87gtaaaaccat cattacaat 19 88 10 DNA Homo sapiens Putative SORY/SOX5.01motif 88 attacaattc 10 89 15 DNA Homo sapiens Putative RPOA/APOLYA.01motif 89 actaaatttg aattg 15 90 12 DNA Homo sapiens PutativeMYT1/MYT1.01 motif 90 taaatttgaa tt 12 91 10 DNA Homo sapiens PutativeOCT1/OCT1.02 motif 91 gatggaaata 10 92 14 DNA Homo sapiens PutativeRREB/RREB1.01 motif 92 ccccaaaaat cccc 14 93 8 DNA Homo sapiens PutativeMZF1/MZF1.01 motif 93 cgagggga 8 94 9 DNA Homo sapiens PutativePCAT/ACAAT.01 motif 94 cccccaatt 9 95 21 DNA Homo sapiens PutativeSTAT/STAT3.01 motif 95 cccaatttca ggcaactact g 21 96 24 DNA Homo sapiensPutative GFI1/GFI1.01 motif 96 aagacagaaa tcagaccagt agtt 24 97 15 DNAHomo sapiens Putative 1RFF/ISRE.01 motif 97 cagaaaagga aagta 15 98 12DNA Homo sapiens Putative NFAT/NFAT.01 motif 98 aaaaggaaag ta 12 99 14DNA Homo sapiens Putative SRFF/SRF.02 motif 99 gtccagaaaa ggaa 14 100 10DNA Homo sapiens Putative RPOA/DTYPEPA.01 motif 100 tacattaaat 10 101 15DNA Homo sapiens Putative OCTP/OCT1P.01 motif 101 ctccatatac attaa 15102 22 DNA Homo sapiens Putative XSEC/STAF.01 motif 102 gctaccccagatgccaaaga ct 22 103 16 DNA Homo sapiens Putative LYMF/TH1E47.01 motif103 tttggcatct ggggta 16 104 30 DNA Homo sapiens Putative HOXF/HOX1-3.01motif 104 agcaagtacg aatattagtc taccacctca 30 105 15 DNA Homo sapiensPutative OCTP/OCT1P.01 motif 105 actaatattc gtact 15 106 19 DNA Homosapiens Putative SEF1/SEF1.01 motif 106 tttatgtgca tctgaggtg 19 107 19DNA Homo sapiens Putative CDXF/CDX2.01 motif 107 taatattttt atgtgcatc 19108 14 DNA Homo sapiens Putative OCT1/OCT1.05 motif 108 aatattttta tgtg14 109 14 DNA Homo sapiens Putative OCT1/OCT1.05 motif 109 aaatattacatatc 14 110 12 DNA Homo sapiens Putative CREB/E4BP4.01 motif 110agatatgtaa ta 12 111 14 DNA Homo sapiens Putative GATA/GATA.01 motif 111agatatgtaa taat 14 112 10 DNA Homo sapiens Putative VBPF/VBP.01 motif112 attacatatc 10 113 15 DNA Homo sapiens Putative EVI1/EVI1.03 motif113 agaaaagaaa agata 15 114 12 DNA Homo sapiens Putative NFAT/NFAT.01motif 114 ggaaggaaaa ga 12 115 15 DNA Homo sapiens Putative ETSF/ETS1.01motif 115 gaaggaagta gagag 15 116 20 DNA Homo sapiens PutativeYY1F/YY1.01 motif 116 gtggcaccat cttggctcag 20 117 18 DNA Homo sapiensPutative MYOF/NF1.01 motif 117 tcttggctca gcgcaacc 18 118 17 DNA Homosapiens Putative XBBF/RFX1.01 motif 118 ttggctcagc gcaacct 17 119 11 DNAHomo sapiens Putative AP1F/NFE2.01 motif 119 ttggctcagc g 11 120 24 DNAHomo sapiens Putative BRAC/BRACH.01 motif 120 agcctctcaa gtagctgaga ttac24 121 14 DNA Homo sapiens Putative TTFF/TTF1.01 motif 121 cctctcaagtagct 14 122 28 DNA Homo sapiens Putative AP1F/BEL1.01 motif 122tggtgcgtgc ctgtaatctc agctactt 28 123 10 DNA Homo sapiens PutativeGATA/GATA3.01 motif 123 tgagattaca 10 124 16 DNA Homo sapiens PutativeAHRR/AHRARNT.01 motif 124 gtagtggtgc gtgcct 16 125 16 DNA Homo sapiensPutative MEF2/HMEF2.01 motif 125 atataaaaat tagcca 16 126 17 DNA Homosapiens Putative HNF1/HNF1.02 motif 126 ggctaatttt tatattt 17 127 15 DNAHomo sapiens Putative TBPF/TATA.01 motif 127 atataaaaat tagcc 15 128 14DNA Homo sapiens Putative FKHD/XFD2.01 motif 128 aatataaaaa ttag 14 12914 DNA Homo sapiens Putative OCT1/OCT1.05 motif 129 ctaattttta tatt 14130 17 DNA Homo sapiens Putative MEF2/RSRFC4.02 motif 130 ctactaaaaatataaaa 17 131 9 DNA Homo sapiens Putative GATA/LMO2COM.02 motif 131gagataggg 9 132 9 DNA Homo sapiens Putative AREB/AREB6.04 motif 132gggtttcac 9 133 10 DNA Homo sapiens Putative CREB/HLF.01 motif 133gtttcaccat 10 134 16 DNA Homo sapiens Putative ARP1/ARP1.01 motif 134tgaactcctg acctca 16 135 16 DNA Homo sapiens Putative T3RH/T3R.01 motif135 gtttgaggtc aggagt 16 136 10 DNA Homo sapiens Putative RARF/RAR.01motif 136 aggtcaggag 10 137 13 DNA Homo sapiens Putative RORA/RORA1.01motif 137 cgtttgaggt cag 13 138 8 DNA Homo sapiens PutativeCREB/CREBP1CJUN.01 motif 138 tgacctca 8 139 9 DNA Homo sapiens PutativeLYMF/LYF1.01 motif 139 tttgggagg 9 140 32 DNA Homo sapiens PutativeHOBO/HOGNESS.01 motif 140 ggcggtggct cacgcctgta atcccagcac tt 32 141 12DNA Homo sapiens Putative IKRS/IK2.01 motif 141 tgctgggatt ac 12 142 15DNA Homo sapiens Putative CREB/TAXCREB.01 motif 142 ggtggctcac gcctg 15143 13 DNA Homo sapiens Putative SP1F/SP1.01 motif 143 ccagggcggt ggc 13144 16 DNA Homo sapiens Putative FKHD/FREAC2.01 motif 144 agaaagtaaagaggcc 16 145 17 DNA Homo sapiens Putative TBPF/MTATA.01 motif 145ttctttaaac ccagttc 17 146 10 DNA Homo sapiens Putative MEF2/MEF2.05motif 146 ggtttaaaga 10 147 18 DNA Homo sapiens Putative XBBF/MIFI.01motif 147 ggggtgtacg gaaaccta 18 148 9 DNA Homo sapiens PutativeAREB/AREB6.04 motif 148 aggtttccg 9 149 8 DNA Homo sapiens PutativeE2FF/E2F.02 motif 149 gcccgaaa 8 150 16 DNA Homo sapiens PutativeLYMF/TH1E47.01 motif 150 actggggtct ggagag 16 151 8 DNA Homo sapiensPutative MZF1/MFZF1.01 motif 151 agagggga 8 152 10 DNA Homo sapiensPutative OCT1/OCT1.02 motif 152 catgcaaaac 10 153 24 DNA Homo sapiensPutative PAX5/PAX9.01 motif 153 ggtacccatt gaagtaaggg ccat 24 154 10 DNAHomo sapiens Putative RPOA/DTYPEPA.01 motif 154 cccattgaag 10 155 10 DNAHomo sapiens Putative VBPF/VBP.01 motif 155 cttacttcaa 10 156 8 DNA Homosapiens Putative CREB/CREBP1.01 motif 156 ttacttca 8 157 8 DNA Homosapiens Putative RPOA/LPOLYA.01 motif 157 aaataaat 8 158 17 DNA Homosapiens Putative XBBF/RFX1.01 motif 158 tttcagccca gcaacat 17 159 30 DNAHomo sapiens Putative HOXF/HOX1-3.01 motif 159 cactgatacc ctcattatcaaatggttctt 30 160 13 DNA Homo sapiens Putative GATA/GATA1.03 motif 160atttgataat gag 13 161 13 DNA Homo sapiens Putative IKRS/IK3.01 motif 161tctagggaac agt 13 162 12 DNA Homo sapiens Putative NFAT/NFAT.01 motif162 cattggaaac ag 12 163 9 DNA Homo sapiens Putative AREB/AREB6.04 motif163 ctgtttcca 9 164 11 DNA Homo sapiens Putative ECAT/NFY.02 motif 164tttccaatga c 11 165 18 DNA Homo sapiens Putative CEBP/CEBP.02 motif 165ggactttggg aacctccc 18 166 10 DNA Homo sapiens Putative NFKB/CREL.01motif 166 gggaggttcc 10 167 12 DNA Homo sapiens Putative IKRS/IK2.01motif 167 ctttgggaac ct 12 168 22 DNA Homo sapiens Putative XSEC/STAF.01motif 168 ggttcccaaa gtccagtagg tg 22 169 8 DNA Homo sapiens PutativeSMAD/SMAD3.01 motif 169 gtctgggt 8 170 11 DNA Homo sapiens PutativeCP2F/CP2.01 motif 170 gcagcaccca g 11 171 21 DNA Homo sapiens PutativePAX6/PAX6.01 motif 171 aggactcaag cctcagtccc t 21 172 16 DNA Homosapiens Putative ARP1/ARP1.01 motif 172 tgagtccttg atgctc 16 173 9 DNAHomo sapiens Putative RPAD/PADS.01 motif 173 ggtggtctt 9 174 16 DNA Homosapiens Putative ECAT/NFY.01 motif 174 tcctcccaat ctgggg 16 175 14 DNAHomo sapiens Putative SRFF/SRF.02 motif 175 ccccagattg ggag 14 176 13DNA Homo sapiens Putative SP1F/SP1.01 motif 176 tgggggcggg gga 13 177 12DNA Homo sapiens Putative EGRF/EGR1.01 motif 177 gggcggggga gt 12 178 11DNA Homo sapiens Putative AP1F/AP1.03 motif 178 agtgactccc c 11 179 18DNA Homo sapiens Putative CMYB/CMYB.01 motif 179 tttcacaaca gttggagg 18180 9 DNA Homo sapiens Putative VMYB/VMYB.02 motif 180 tccaactgt 9 18114 DNA Homo sapiens Putative CEBP/CEBPB.01 motif 181 ctgttgtgaa agcc 14182 21 DNA Homo sapiens Putative MINI/MUSCLE_INI.02 motif 182 cctccaccccacccagctct g 21 183 11 DNA Homo sapiens Putative EBOX/SREBP1.02 motif183 ctccacccca c 11 184 24 DNA Homo sapiens Putative PAX5/PAX9.01 motif184 aagagccaga gctgggtggg gtgg 24 185 14 DNA Homo sapiens PutativeSP1F/GC.01 motif 185 gctgggtggg gtgg 14 186 10 DNA Homo sapiens PutativeNFKB/CREL.01 motif 186 tggctcttcc 10 187 12 DNA Homo sapiens PutativeETSF/GABP.01 motif 187 ggaggaagag cc 12 188 19 DNA Homo sapiens PutativeSEF1/SEF1.01 motif 188 ctccaggaca tctggggta 19 189 16 DNA Homo sapiensPutative AP4R/TALIALPHAE47.01 motif 189 taccccagat gtcctg 16 190 18 DNAHomo sapiens Putative REOA/POLYA.01 motif 190 caatacatcc atgatcta 18 19111 DNA Homo sapiens Putative EVI1/EVI1.02 motif 191 agacaagaag a 11 19218 DNA Homo sapiens Putative CMYB/CMYB.01 motif 192 tctaagagct gttgccag18 193 17 DNA Homo sapiens Putative XBBF/RFX1.01 motif 193 tggactcctggcaacag 17 194 18 DNA Homo sapiens Putative MYOF/NF1.01 motif 194cgttggctgg actcctgg 18 195 12 DNA Homo sapiens Putative EGRF/EGR3.01motif 195 gagcgttggc tg 12 196 22 DNA Homo sapiens Putative NOLF/OLF1.01motif 196 aacgagtccc tttgggcttc ct 22 197 9 DNA Homo sapiens PutativeAREB/AREB6.04 motif 197 ctgtttgga 9 198 27 DNA Homo sapiens PutativeGREF/ARE.01 motif 198 gtttgatgtt ccttgtgttc cctttcc 27 199 13 DNA Homosapiens Putative IRFF/IRF2.01 motif 199 ggaaagggaa cac 13 200 16 DNAHomo sapiens Putative LDPS/LDSPOLYA.01 motif 200 tccttgtgtt cccttt 16201 18 DNA Homo sapiens Putative XBBF/RFX1.02 motif 201 agggaacacaaggaacat 18 202 10 DNA Homo sapiens Putative RPOA/DTYPEPA.01 motif 202aacatcaaac 10 203 13 DNA Homo sapiens Putative IKRS/IK1.01 motif 203gtgtgggaag gtt 13 204 21 DNA Homo sapiens Putative XSEC/STAF.02 motif204 ccttcccaca ctgctctaca t 21 205 10 DNA Homo sapiens PutativeRPOA/DTYPEPA.01 motif 205 accacaaaac 10 206 6 DNA Homo sapiens PutativeHAML/AML1.01 motif 206 tgtggt 6 207 6 DNA Homo sapiens PutativeHAML/AML1.01 motif 207 tgtggt 6 208 14 DNA Homo sapiens PutativeECAT/NFY.03 motif 208 atcaacaaat cagc 14 209 10 DNA Homo sapiensPutative TBPF/ATATA.01 motif 209 ttatttcagt 10 210 13 DNA Homo sapiensPutative IRFF/IRF1.01 motif 210 aaaaactgaa ata 13 211 10 DNA Homosapiens Putative VMYB/VMYB.01 motif 211 aaaaactgaa 10 212 21 DNA Homosapiens Putative PAX6/PAX6.01 motif 212 agttttttcg ctgcatttag a 21 213 8DNA Homo sapiens Putative E2FF/E2F.02 motif 213 gcgaaaaa 8 214 23 DNAHomo sapiens Putative PAX5/PAX9.01 motif 214 tctacccatg gaagtgtcag gaa23 215 15 DNA Homo sapiens Putative MTF1/MTF-1.01 motif 215 tcctgcacacttcca 15 216 14 DNA Homo sapiens Putative ETSF/ETS2.01 motif 216tgcaggaaga tgga 14 217 13 DNA Homo sapiens Putative ZFIA/ZID.01 motif217 tgactccatc ttc 13 218 11 DNA Homo sapiens Putative AP1F/AP1FJ.01motif 218 ggtgactcca t 11 219 9 DNA Homo sapiens Putative VMYB/VMYB.02motif 219 ccaaacggg 9 220 16 DNA Homo sapiens Putative ETSF/ELK1.01motif 220 caaacgggat gatcca 16 221 12 DNA Homo sapiens PutativeNFKB/NFKAPPAB.02 motif 221 cgggatgatc ca 12 222 9 DNA Homo sapiensPutative AREB/AREB6.04 motif 222 ctgtttctt 9 223 13 DNA Homo sapiensPutative ZFI1A/ZID.01 motif 223 cggctctaac aca 13 224 18 DNA Homosapiens Putative XBBF/RFX1.02 motif 224 ctctaacaca agcaacag 18 225 18DNA Homo sapiens Putative CMYB/CMYB.01 motif 225 gtttgttgct gttgcttg 18226 15 DNA Homo sapiens Putative CREB/TAXCREB.02 motif 226 gaggaaatacgtctt 15 227 14 DNA Homo sapiens Putative ETSF/ETS2.01 motif 227aagaggaaat acgt 14 228 12 DNA Homo sapiens Putative NFAT/NFAT.01 motif228 aagaggaaat ac 12 229 11 DNA Homo sapiens Putative EVI1/EVI1.02 motif229 tgagaagatt a 11 230 16 DNA Homo sapiens Putative OAZF/ROAZ.01 motif230 cagcatcctt aggtga 16 231 11 DNA Homo sapiens PutativeEBOR/DELTAEF1.01 motif 231 cctcacctaa g 11 232 8 DNA Homo sapiensPutative CREB/CREBP1.01 motif 232 tcacctaa 8 233 15 DNA Homo sapiensPutative HNF4/HNF4.02 motif 233 tgggtccaga ggcct 15 234 11 DNA Homosapiens Putative GATA/GATA.01 motif 234 agataaggcc t 11 235 12 DNA Homosapiens Putative CREB/E4BP4.01 motif 235 ccttatctaa aa 12 236 10 DNAHomo sapiens Putative TBPF/ATATA.01 motif 236 ttagataagg 10 237 18 DNAHomo sapiens Putative XBBF/MIF1.01 motif 237 acggtgccca gccaccca 18 2388 DNA Homo sapiens Putative EBOX/USF.02 motif 238 acacatgt 8 239 10 DNAHomo sapiens Putative VBPF/VBP.01 motif 239 attacatgtg 10 240 12 DNAHomo sapiens Putative IKRS/IK2.01 motif 240 tgctgggatt ac 12 241 21 DNAHomo sapiens Putative NRSF/NRSF.01 motif 241 cccagcactt tggaaggccg a 21242 19 DNA Homo sapiens Putative TANT/TANTIGEN.01 motif 242 ggaaggccgaggcaggtgg 19 243 13 DNA Homo sapiens Putative AREB/AREB6.01 motif 243gatccacctg cct 13 244 10 DNA Homo sapiens Putative MYOD/MYOD.02 motif244 tccacctgcc 10 245 11 DNA Homo sapiens Putative EBOX/SREBP1.02 motif245 gatcacccga g 11 246 10 DNA Homo sapiens Putative RARF/RAR.01 motif246 aggtcaggag 10 247 10 DNA Homo sapiens Putative CREB/HLF.01 motif 247gtttcgccat 10 248 10 DNA Homo sapiens Putative CLOX/CDPCR3HD.01 motif248 tattgatgag 10 249 10 DNA Homo sapiens Putative OCT1/OCT1.02 motif249 aatgcaaaaa 10 250 12 DNA Homo sapiens Putative MYT1/MYT1.01 motif250 aaaaattagc tt 12 251 6 DNA Homo sapiens Putative HAML/AML1.01 motif251 tgtggt 6 252 12 DNA Homo sapiens Putative IKRS/IK2.01 motif 252ggctgggatt ac 12 253 19 DNA Homo sapiens Putative AHRR/AHRARNT.02 motif253 tgggtttgag tgattctcc 19 254 13 DNA Homo sapiens PutativeCHOP/CHOP.01 motif 254 cactgcaatc tcc 13 255 19 DNA Homo sapiensPutative OCT1/OCT1.01motif 255 gagattatgc cactgcact 19 256 16 DNA Homosapiens Putative MEF2/MEF2.01 motif 256 ctcaaaaaat aaaata 16 257 19 DNAHomo sapiens Putative CDXF/CDX2.01 motif 257 caaaggtttt attttattt 19 25811 DNA Homo sapiens Putative EVI1/EVI1.03 motif 258 aaataaaata a 11 25918 DNA Homo sapiens Putative RPOA/POLYA.01 motif 259 aaataaaacc tttggggc18 260 8 DNA Homo sapiens Putative E2FF/E2F.02 motif 260 gccccaaa 8 26122 DNA Homo sapiens Putative XSEC/STAF.01 motif 261 aatccccagaattctggact ct 22 262 12 DNA Homo sapiens Putative NFKB/NFKAPPAB.02 motif262 ggggattttc aa 12 263 17 DNA Homo sapiens Putative HNF1/HNF1.02 motif263 ggctattcaa taaatgg 17 264 8 DNA Homo sapiens Putative RPOA/LPOLYA.01motif 264 caataaat 8 265 15 DNA Homo sapiens Putative TBPF/TATA.01 motif265 atataaatcc cattt 15 266 9 DNA Homo sapiens Putative HMTB/MTBF.01motif 266 tgggattta 9 267 10 DNA Homo sapiens Putative CREB/HLF.01 motif267 gttatgtgat 10 268 10 DNA Homo sapiens Putative VBPF/VBP.01 motif 268gttatgtgat 10 269 12 DNA Homo sapiens Putative CREB/CREB.03 motif 269tctgacgcag tt 12 270 14 DNA Homo sapiens Putative GATA/GATA1.01 motif270 tagttgatag gaga 14 271 15 DNA Homo sapiens Putative CLOX/CLOX.01motif 271 aaaatcgaat agttg 15 272 12 DNA Homo sapiens PutativeNFAT/NFAT.01 motif 272 tgaaggaaaa tc 12 273 24 DNA Homo sapiens PutativeGFI1/GFI1.01 motif 273 aatttaaaaa tcacatcaag ggat 24 274 10 DNA Homosapiens Putative MEF2/MEF2.05 motif 274 aatttaaaaa 10 275 10 DNA Homosapiens Putative GATA/GATA3.02 motif 275 agggatctaa 10 276 16 DNA Homosapiens Putative FKHD/FREAC3.01 motif 276 gggatctaaa taaaga 16 277 10DNA Homo sapiens Putative MEF2/MEF2.05 motif 277 gatctaaata 10 278 8 DNAHomo sapiens Putative RPOA/LPOLYA.01 motif 278 aaataaag 8 279 9 DNA Homosapiens Putative HMTB/MTBF.01 motif 279 agctattta 9 280 9 DNA Homosapiens Putative VMYB/VMYB.02 motif 280 cccaactga 9 281 8 DNA Homosapiens Putative SMAD/SMAD3.01 motif 281 gtctggtc 8 282 15 DNA Homosapiens Putative HNF4/HNF4.02 motif 282 aaggaccaaa cctct 15 283 11 DNAHomo sapiens Putative MYT1/MYT1.02 motif 283 agaaagttct a 11 284 10 DNAHomo sapiens Putative HEAT/HSF1.01 motif 284 agaaagttct 10 285 8 DNAHomo sapiens Putative MZF1/MZF1.01 motif 285 aatgggga 8 286 10 DNA Homosapiens Putative TBPF/TATA.02 motif 286 tctgtaaaat 10 287 13 DNA Homosapiens Putative GATA/GATA1.03 motif 287 tacagataaa ggg 13 288 16 DNAHomo sapiens Putative ETSF/PU1.01 motif 288 gaatgaggaa gggtaa 16 289 10DNA Homo sapiens Putative CREB/HLF.01 motif 289 gttacttcat 10 290 10 DNAHomo sapiens Putative VBPF/VBP.01 motif 290 gttacttcat 10 291 13 DNAHomo sapiens Putative RORA/RORA2.01 motif 291 gtaacttggt caa 13 292 16DNA Homo sapiens Putative LDPS/LDSPOLYA.01 motif 292 ggagtgtgtg tgcatg16 293 8 DNA Homo sapiens Putative EBOX/USF.02 motif 293 acacatgc 8 29410 DNA Homo sapiens Putative NFKB/NFKAPPAB.01 motif 294 gggggtgccc 10295 21 DNA Homo sapiens Putative MINI/MUSCLE_INI.03 motif 295 ggcaccccccaccccgaccc c 21 296 21 DNA Homo sapiens Putative REBV/EBVR.01 motif 296ggggtcgggg tggggggtgc c 21 297 13 DNA Homo sapiens Putative EGRF/WT1.01motif 297 gggtgggggg tgc 13 298 14 DNA Homo sapiens Putative SP1F/GC.01motif 298 tcggggtggg gggt 14 299 14 DNA Homo sapiens PutativeRREB/RREB1.01 motif 299 ccccaccccg accc 14 300 9 DNA Homo sapiensPutative PCAT/ACAAT.01 motif 300 ccaccactg 9 301 16 DNA Homo sapiensPutative ARP1/ARP1.01 motif 301 tgattccttg ctctca 16 302 11 DNA Homosapiens Putative MYT1/MYT1.02 motif 302 tcaaagttgt t 11 303 15 DNA Homosapiens Putative IRFF/ISRE.01 motif 303 ctgtaccaga aactc 15 304 13 DNAHomo sapiens Putative EGRF/WT1.01 motif 304 gtgtgggagg ctc 13 305 10 DNAHomo sapiens Putative RARF/RAR.01 motif 305 aggtcaccca 10 306 13 DNAHomo sapiens Putative RORA/RORA1.01 motif 306 agaagaaggt cac 13 307 16DNA Homo sapiens Putative EVI1/EVI1.01 motif 307 agccaagaga agaagg 16308 14 DNA Homo sapiens Putative OCT1/OCT1.05 motif 308 ctcattttaa ttca14 309 15 DNA Homo sapiens Putative OCTB/TST1.01 motif 309 agtgaattaaaatga 15 310 13 DNA Homo sapiens Putative RBIT/BRIGHT.01 motif 310agtgaattaa aat 13 311 8 DNA Homo sapiens Putative NKXH/NKX25.02 motif311 tttaattc 8 312 27 DNA Homo sapiens Putative GREF/PRE.01 motif 312ttcatagtgt tgttttgttc tcgtttt 27 313 18 DNA Homo sapiens PutativeRPOA/POLYA.01 motif 313 gaacaaaaca acactatg 18 314 18 DNA Homo sapiensPutative AHRR/AHR.01 motif 314 actccagctt gggtgaga 18 315 24 DNA Homosapiens Putative GFI1/GFI1.01 motif 315 agtgctgcaa tcacagctca ttgc 24316 9 DNA Homo sapiens Putative LYMF/LYF1.01 motif 316 tttgggagg 9 31732 DNA Homo sapiens Putative HOBO/HOGNESS.01 motif 317 cacggtggctcacacctgta atcccagcac tt 32 318 12 DNA Homo sapiens Putative IKRS/IK2.01motif 318 tgctgggatt ac 12 319 16 DNA Homo sapiens Putative MYOD/E47.02motif 319 gattacaggt gtgagc 16 320 12 DNA Homo sapiens PutativeAREB/AREB6.02 motif 320 tcacacctgt aa 12 321 12 DNA Homo sapiensPutative BRAC/TBX5.01 motif 321 acaggtgtga gc 12 322 17 DNA Homo sapiensPutative TBPF/MTATA.01 motif 322 ctgtttaaaa ccctata 17 323 16 DNA Homosapiens Putative FKHD/FREAC2.01 motif 323 gggttttaaa cagtaa 16 324 10DNA Homo sapiens Putative MEF2/MEF2.05 motif 324 gttttaaaca 10 325 18DNA Homo sapiens Putative CEBP/CEBP.02 motif 325 tgcctgcggt aagtcgta 18326 22 DNA Homo sapiens Putative NOLF/OLF1.01 motif 326 aaagggtccccccggggcct gt 22 327 12 DNA Homo sapiens Putative AP2F/AP2.01 motif 327gtccccccgg gg 12 328 8 DNA Homo sapiens Putative MZF1/MZF1.01 motif 328cgggggga 8 329 22 DNA Homo sapiens Putative HEN1/HEN1.01 motif 329ccagggtaca gctgtgacac cg 22 330 10 DNA Homo sapiens Putative AP4R/AP4.01motif 330 cacagctgta 10 331 14 DNA Homo sapiens Putative GATA/GATA1.02motif 331 actgggataa tcca 14 332 12 DNA Homo sapiens PutativeNFKB/NFKAPPAB.02 motif 332 tgggataatc ca 12 333 13 DNA Homo sapiensPutative FKHD/HFH8.01 motif 333 tagataaaca aaa 13 334 11 DNA Homosapiens Putative GATA/GATA.01 motif 334 agtaaaacaa a 11 335 12 DNA Homosapiens Putative SORY/SRY.01 motif 335 ataaacaaaa at 12 336 12 DNA Homosapiens Putative CREB/CREB.02 motif 336 ggaatgacga tc 12 337 13 DNA Homosapiens Putative PAX3/PAX3.01 motif 337 tcgtcattcc att 13 338 12 DNAHomo sapiens Putative TEAF/TEF1.01 motif 338 gtcattccat tt 12 339 18 DNAHomo sapiens Putative PAX1/PAX1.01 motif 339 ccatttctct ctgtatat 18 34012 DNA Homo sapiens Putative NFAT/NFAT.01 motif 340 gcttggaaaa at 12 34115 DNA Homo sapiens Putative BARB/BARBIE.01 motif 341 atgaaaaggg cttgg15 342 10 DNA Homo sapiens Putative OCT1/OCT1.02 motif 342 catgaaaagg 10343 22 DNA Homo sapiens Putative AP1F/TCF11MAFG.01 motif 343 ttttcatgaatgatcagtta tt 22 344 10 DNA Homo sapiens Putative PITI1/PIT1.01 motif344 gatcattcat 10 345 10 DNA Homo sapiens Putative VMYB/VMYB.01 motif345 aataactgat 10 346 14 DNA Homo sapiens Putative ETSF/ETS2.01 motif346 tgcaggaaat aact 14 347 24 DNA Homo sapiens Putative GFI1/GFI1.01motif 347 aaaaaaaaaa tcagtgcagg aaat 24 348 11 DNA Homo sapiens PutativeAP1F/AP1FJ.01 motif 348 ggtgacagag t 11 349 11 DNA Homo sapiens PutativeEBOX/SREBP1.02 motif 349 gatcatgcca c 11 350 13 DNA Homo sapiensPutative PAX3/PAX3.01 motif 350 tcggctcgct gca 13 351 10 DNA Homosapiens Putative HEAT/HSF1.01 motif 351 agaagaatcg 10 352 21 DNA Homosapiens Putative XSEC/STAF.02 motif 352 gagtaccatc atgcccggct a 21 35320 DNA Homo sapiens Putative P53F/P53.01 motif 353 catcatgccc ggctaatttt20 354 17 DNA Homo sapiens Putative MEF2/RSRFC4.02 motif 354 ctactaaaaatacaaaa 17 355 18 DNA Homo sapiens Putative SRFF/SRF.01 motif 355ttcaccatat tggccagg 18 356 11 DNA Homo sapiens Putative ECAT/NFY.02motif 356 tggccaatat g 11 357 15 DNA Homo sapiens Putative HNF4/HNF4.02motif 357 cagatcgcaa ggtcc 15 358 9 DNA Homo sapiens PutativeLYMF/LYF1.01 motif 358 tttgggagg 9 359 32 DNA Homo sapiens PutativeHOBO/HOGNESS.01 motif 359 cgcggtggct cacgcctgta atcccagcac tt 32 360 12DNA Homo sapiens Putative IKRS/IK2.01 motif 360 tgctgggatt ac 12 361 15DNA Homo sapiens Putative CREB/TAXCREB.01 motif 361 ggtggctcac gcctg 15362 10 DNA Homo sapiens Putative EBOX/MYCMAX.03 motif 362 gccaggcgcg 10363 10 DNA Homo sapiens Putative GATA/GATA3.02 motif 363 actgatataa 10364 15 DNA Homo sapiens Putative EVI1/EVI1.04 motif 364 tgatataaaa agaat15 365 10 DNA Homo sapiens Putative MEF2/MEF2.05 motif 365 gatataaaaa 10366 15 DNA Homo sapiens Putative TBPF/TATA.01 motif 366 atataaaaag aattt15 367 15 DNA Homo sapiens Putative RPOA/APOLYA.01 motif 367 aaaaaaattcttttt 15 368 10 DNA Homo sapiens Putative MEF2/MEF2.05 motif 368aatttaaaaa 10 369 11 DNA Homo sapiens Putative EBOX/SREBP1.02 motif 369tttctcccca c 11 370 8 DNA Homo sapiens Putative MZF1/MZF1.01 motif 370agtgggga 8 371 21 DNA Homo sapiens Putative MINI/MUSCLE_INI.03 motif 371ccccactccc acccccaggc t 21 372 14 DNA Homo sapiens PutativeRREB/RREB1.01 motif 372 ccccactccc accc 14 373 13 DNA Homo sapiensPutative EGRF/WT1.01 motif 373 gggtgggagt ggg 13 374 12 DNA Homo sapiensPutative AP2F/AP2.01 motif 374 cacccccagg ct 12 375 17 DNA Homo sapiensPutative TBPF/MTATA.01 motif 375 ccttataaag cagcctc 17 376 6 DNA Homosapiens Putative HAML/AMLI.01 motif 376 tgtggt 6 377 14 DNA Homo sapiensPutative ETSF/ELK1.02 motif 377 gggcccggaa ttgg 14 378 16 DNA Homosapiens Putative LYMF/THIE47.01 motif 378 aattgggtct ggggca 16 379 28DNA Homo sapiens Putative PAX5/PAX5.01 motif 379 cccaagagca gggcagagaagcaagcaa 28 380 23 DNA Homo sapiens Putative LTUP/TAACC.01 motif 380tgcccctgag gctaacccca aga 23 381 28 DNA Homo sapiens PutativePAX5/PAX5.01 motif 381 ctcaggggca gggttgagag tcaggctt 28 382 25 DNA Homosapiens Putative PCAT/CLTR_CAAT.01 motif 382 gccaagcctg actctcaacc ctgcc25 383 12 DNA Homo sapiens Putative MYOD/MYF5.01 motif 383 aggcagcagg ag12 384 16 DNA Homo sapiens Putative ETSF/ELK1.01 motif 384 gcagcaggaggtccag 16 385 8 DNA Homo sapiens Putative SMAD/SMAD3.01 motif 385gtctggac 8 386 10 DNA Homo sapiens Putative GATA/GATA2.02 motif 386ggagatacca 10 387 9 DNA Homo sapiens Putative HMTB/MTBF.01 motif 387tggtatctc 9 388 13 DNA Homo sapiens Putative EGRF/WT1.01 motif 388gagagggcgc atc 13 389 20 DNA Homo sapiens Putative PERO/PPARA.01 motif389 ctgaaacagg aaaaaggcag 20 390 14 DNA Homo sapiens PutativeGKLF/GKLF.01 motif 390 aaacaggaaa aagg 14 391 12 DNA Homo sapiensPutative NFAT/NFAT.01 motif 391 aacaggaaaa ag 12 392 9 DNA Homo sapiensPutative AREB/AREB6.04 motif 392 ctgtttcag 9 393 12 DNA Homo sapiensPutative SORY/SRY.01 motif 393 aaaaacaaaa ca 12 394 12 DNA Homo sapiensPutative FKHD/HFH2.01 motif 394 aaaaaaacaa aa 12 395 13 DNA Homo sapiensPutative EGRF/WT1.01 motif 395 gagagggagg gag 13 396 13 DNA Homo sapiensPutative EGRF/WT1.01 motif 396 gagagggagg gag 13 397 14 DNA Homo sapiensPutative GKLF/GKLF.01 motif 397 agagagagag aggg 14 398 13 DNA Homosapiens Putative SP1F/SP1.01 motif 398 ggagggaggg gga 13 399 14 DNA Homosapiens Putative GKLF/GKLF.01 motif 399 gaaggaggga gggg 14 400 10 DNAHomo sapiens Putative OCT1/OCT1.02 motif 400 gatgcacata 10 401 9 DNAHomo sapiens Putative EVI1/EVI1.06 motif 401 acaaggtag 9 402 13 DNA Homosapiens Putative TCFF/TCF11.01 motif 402 gtcatcctgc tgt 13 403 21 DNAHomo sapiens Putative MINI/MUSCLE_INI.01 motif 403 tccctcctcc acaccagcaga 21 404 21 DNA Homo sapiens Putative NRSF/NRSF.01 motif 404 ttcagcaacaagaatagccg a 21 405 15 DNA Homo sapiens Putative CLOX/CDPCR3.01 motif405 cagcaacaag aatag 15 406 25 DNA Homo sapiens PutativePCAT/CLTR_CAAT.01 motif 406 cccaagaagc atcctgcagg ctttc 25 407 15 DNAHomo sapiens Putative BARB/BARBIE.01 motif 407 tcaaaaagca gaaag 15 40816 DNA Homo sapiens Putative MEF2/MMEF2.01 motif 408 tgctttaaaa tacact16 409 10 DNA Homo sapiens Putative TBPF/TATA.02 motif 409 gctttaaaat 10410 10 DNA Homo sapiens Putative TBPF/ATATA.01 motif 410 ctatgtatgc 10411 12 DNA Homo sapiens Putative MYT1/MYT1.01 motif 411 catagttaac tg 12412 10 DNA Homo sapiens Putative GATA/GATA3.02 motif 412 ctagatgtta 10413 14 DNA Homo sapiens Putative FKHD/XFD3.01 motif 413 aaggttaaca tcta14 414 12 DNA Homo sapiens Putative MYT1/MYT1.01 motif 414 aaaggttaac at12 415 16 DNA Homo sapiens Putative AP4R/TAL1BETA-E47.01 motif 415aaacacagat ggaggc 16 416 12 DNA Homo sapiens Putative EGRF/EGR1.01 motif416 ttctgtgggc gg 12 417 13 DNA Homo sapiens Putative ZFIA/ZID.01 motif417 cggctccagc ctc 13 418 15 DNA Homo sapiens Putative CREB/TAXCREB.02motif 418 cgggatctgc gggaa 15 419 18 DNA Homo sapiens PutativeCEBP/CEBP.02 motif 419 gatctgcggg aagacacg 18 420 15 DNA Homo sapiensPutative E2FF/E2F.01 motif 420 tctgcgggaa gacac 15 421 12 DNA Homosapiens Putative EBOX/NMYC.01 motif 421 ttccccgtgt ct 12 422 12 DNA Homosapiens Putative CLOX/CDP.01 motif 422 tcattaatca aa 12 423 15 DNA Homosapiens Putative HNF1/HNF1.01 motif 423 gattaatgat ttatt 15 424 18 DNAHomo sapiens Putative CART/CART1.01 motif 424 gatttatttt gattaacg 18 4258 DNA Homo sapiens Putative RPOA/LPOLYA.01 motif 425 aaataaat 8 426 15DNA Homo sapiens Putative HNF1/HNF1.01 motif 426 cgttaatcaa aataa 15 42724 DNA Homo sapiens Putative COMP/COMP1.01 motif 427 tattttgattaacgccgtca cagt 24 428 14 DNA Homo sapiens Putative CREB/ATF.01 motif428 ctgtgacggc gtta 14 429 28 DNA Homo sapiens Putative PAX5/PAX5.02motif 429 agggactgct ctaaggcgtc actgtgac 28 430 21 DNA Homo sapiensPutative PAX6/PAX6.01 motif 430 cacagtgacg ccttagagca g 21 431 14 DNAHomo sapiens Putative CREB/ATF.01 motif 431 cagtgacgcc ttag 14 432 11DNA Homo sapiens Putative WHZF/WHN.01 motif 432 agtgacgcct t 11 433 16DNA Homo sapiens Putative FKHD/FREAC4.01 motif 433 cccgggtgaa caggga 16434 12 DNA Homo sapiens Putative EGRF/NGFIC.01 motif 434 cagcgagggt gg12 435 13 DNA Homo sapiens Putative SP1F/SP1.01 motif 435 tgggggcgga cgc13 436 14 DNA Homo sapiens Putative GKLF/GKLF.01 motif 436 ggaaagaggagggg 14 437 25 DNA Homo sapiens Putative PCAT/CLTR_CAAT.01 motif 437accaaggccc cgcccctcct ctttc 25 438 13 DNA Homo sapiens PutativeSP1F/SP1.01 motif 438 gaggggcggg gcc 13 439 14 DNA Homo sapiens PutativeRREB/RREB1.01 motif 439 ccccacccga ccaa 14 440 12 DNA Homo sapiensPutative TEAF/TEF1.01 motif 440 cccattccat ac 12 441 24 DNA Homo sapiensPutative PAX5/PAX9.01 motif 441 aatgggcagg gtggggggga tggg 24 442 14 DNAHomo sapiens Putative RREB/RREB1.01 motif 442 ccccaccctg ccca 14 443 13DNA Homo sapiens Putative EGRF/WT1.01 motif 443 gggtgggggg gat 13 444 14DNA Homo sapiens Putative RREB/RREB1.01 motif 444 gcccatcccc ccca 14 4458 DNA Homo sapiens Putative MZF1/MZF1.01 motif 445 ggggggga 8 446 13 DNAHomo sapiens Putative SP1F/SP1.01 motif 446 gatgggcggg gta 13 447 13 DNAHomo sapiens Putative SP1F/SP1.01 motif 447 gatgggcggg gcc 13 448 13 DNAHomo sapiens Putative E2FF/E2F.03 motif 448 gcccgggaaa ttc 13 449 22 DNAHomo sapiens Putative NOLF/OLF1.01 motif 449 ggaaattccc cggcgcgggc ag 22450 10 DNA Homo sapiens Putative NFKB/NFKAPPAB.01 motif 450 gggaatttcc10 451 13 DNA Homo sapiens Putative IKRS/IK1.01 motif 451 gccggggaat ttc13 452 22 DNA Homo sapiens Putative HEN1/HEN1.01 motif 452 ctggctgtcagctgagccgc gc 22 453 10 DNA Homo sapiens Putative AP4R/AP4.01 motif 453ctcagctgac 10 454 13 DNA Homo sapiens Putative SP1F/SP1.01 motif 454gctgggcggg gtc 13 455 12 DNA Homo sapiens Putative EGRF/NGFIC.01 motif455 tggcggaggg gg 12 456 12 DNA Homo sapiens Putative EGRF/NGFIC.01motif 456 cggcggtggc gg 12 457 12 DNA Homo sapiens PutativeEGRF/NGFIC.01 motif 457 gggcggcggc gg 12 458 13 DNA Homo sapiensPutative SPIF/SP1.01 motif 458 ggcgggcggc ggc 13 459 12 DNA Homo sapiensPutative AP2F/AP2.01 motif 459 cgcccgccgg ca 12 460 281 DNA Homo sapiensrepeat element 460 cagctctatt gaggtataat ccacatgcca taaaattcaccccatttgta aatgtatgat 60 tcatggcttt caattacact taaaaagttg taaaaccatcattacaattc aaatttagta 120 tatttccatc atcccccaaa aatcccctcg agttcctttgcagttcaaag ccacccccaa 180 tttcaggcaa ctactggtct gatttctgtc tttttctactttccttttct ggacatttaa 240 tgtatatgga gtcatagcat atgtagtctt tggcatctgg g281 461 20 DNA Homo sapiens Short repeat element 461 ttcttttcttttccttcctt 20 462 328 DNA Homo sapiens ALU repeat element 462 tccttcttacctttcttcct tctctctctc tctctctttc tttttggaca gagtctcact 60 ccatggcccaggctggagtg cagtggcacc atcttggctc agcgcaacct ttgactccca 120 ggctcaagcaattctcctgc ctcagcctct caagtagctg agattacagg cacgcaccac 180 tactgcctggctaattttta tatttttagt agagataggg tttcaccatg ttagccaggc 240 tggtcttgaactcctgacct caaacgatcc tcccaaagtg ctgggattac aggcgtgagc 300 caccgccctgggcctcttta ctttcttt 328 463 300 DNA Homo sapiens ALU repeat element 463ctgggcaccg tggctcacac atgtaatccc agcactttgg aaggccgagg caggtggatc 60acccgaggtc aggagttcaa taccaggctg gtcaacatgg cgaaacctca tcaatacgaa 120aaatgcaaaa attagcttgg tgtggtggca cacgcctgta atcccagcca cttgggaggc 180tgaggcagga gaatcactca aacccaggag gtggagattg cagtgagctg agattatgcc 240actgcactcc agcctgggca acagagtgag actccacctc aaaaaataaa ataaaacctt 300

What is claimed is:
 1. An isolated nucleic acid molecule that comprisesat least 20 consecutive nucleotides but not more than 1500 consecutivenucleotides of the sequence of SEQ ID NO:
 1. 2. An isolated nucleic acidmolecule comprising a promoter which comprises at least 20 consecutivenucleotides but not more than 1500 consecutive nucleotides of thesequence of SEQ ID NO: 1, said promoter being operably linked to aheterologous nucleic acid sequence.
 3. The isolated nucleic acidmolecule according to claim 2, where said heterologous nucleic acidsequence is capable of being expressed in ocular tissue.
 4. The isolatednucleic acid molecule according to claim 2, where said heterologousnucleic acid sequence is capable of being expressed in optic nervecells.
 5. The isolated nucleic acid molecule according to claim 2, wheresaid heterologous nucleic acid sequence is capable of being expressed inretinal cells.
 6. The isolated nucleic acid molecule according to claim2, where said heterologous nucleic acid sequence is capable of beingexpressed in trabecular meshwork cells.
 7. The isolated nucleic acidmolecule according to claim 2, where said heterologous nucleic acidsequence is selected from the group consisting of a coding sequence, atoxin, and a reporter gene.
 8. The isolated nucleic acid moleculeaccording to claim 7, wherein the reporter gene is selected from thegroup consisting of green fluorescent protein and luciferase.
 9. Theisolated nucleic acid molecule according to claim 2, where saidheterologous nucleic acid sequence is capable of being transcribed as anantisense RNA.
 10. The isolated nucleic acid molecule according to claim9, wherein said antisense RNA is capable of binding to a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO: 1 or complementsthereof under physiological conditions.
 11. The isolated nucleic acidmolecule according to claim 10, wherein said antisense RNA is capable ofbinding to a nucleic acid molecule having a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 3 through 463 and complementsthereof under physiological conditions.
 12. A nucleic acid moleculecapable of detecting a single nucleotide polymorphism selected fromtable
 1. 13. The nucleic acid molecule according to claim 12, whereinthe nucleic acid molecule is capable of detecting a single nucleotidepolymorphism selected from table
 4. 14. The nucleic acid moleculeaccording to claim 12, wherein said nucleic acid molecule is capable ofdetecting a guanine.
 15. The nucleic acid molecule according to claim12, wherein said nucleic acid molecule is capable of detecting acytosine.
 16. The nucleic acid molecule according to claim 12, whereinsaid nucleic acid molecule is capable of detecting a thymine.
 17. Thenucleic acid molecule according to claim 12, wherein said nucleic acidmolecule is capable of detecting an adenine.
 18. The nucleic acidmolecule according to claim 12, wherein said nucleic acid molecule doesnot specifically hybridize to a nucleic acid molecule consisting of SEQID NO:
 1. 19. A nucleic acid molecule capable of detecting a singlenucleotide polymorphism in an optineurin promoter by specificallydetecting said single nucleotide polymorphism in said optineurinpromoter, wherein said nucleic acid molecule does not specificallyhybridize to a nucleic acid molecule consisting of SEQ ID NO:
 1. 20. Ahost cell comprising a nucleic acid molecule comprising a promoter whichcomprises at least 20 consecutive nucleotides but not more than 1500consecutive nucleotides of the sequence of SEQ ID NO: 1, said promoterbeing operably linked to a heterologous nucleic acid sequence.
 21. Thehost cell of claim 20, wherein said host cell is selected from the groupconsisting of a non-human mammalian cell, a bacterial cell, and anisolated human cell.
 22. A method for diagnosing glaucoma in a sampleobtained from a cell or a bodily fluid by detecting a polymorphism in apromoter region of the optineurin gene, comprising the steps of: (A)incubating under conditions permitting nucleic acid hybridization, amarker nucleic acid molecule, said marker nucleic acid molecule having anucleic acid sequence that specifically hybridizes to a sequenceselected from the group consisting of SEQ ID NO: 1 and a complementthereof, and a complementary nucleic acid molecule obtained from asample, wherein nucleic acid hybridization between said marker nucleicacid molecule and said complementary nucleic acid molecule permits thedetection of said polymorphism; (B) permitting hybridization betweensaid marker nucleic acid molecule and said complementary nucleic acidmolecule; and (C) detecting the presence of said polymorphism, whereinthe detection of said polymorphism is diagnostic of glaucoma.
 23. Themethod for diagnosing glaucoma of claim 22, wherein said polymorphism isa single nucleotide polymorphism.
 24. The method for diagnosing glaucomaof claim 22, wherein said marker nucleic acid molecule has a nucleotidesequence selected from the group consisting of SEQ ID NOs: 3 through463.
 25. The method for diagnosing glaucoma of claim 22, furthercomprising a second marker nucleic acid molecule.
 26. The method fordiagnosing glaucoma of claim 22, wherein the cell or bodily fluidcomprises ocular tissue.
 27. The method for diagnosing glaucoma of claim22, wherein the cell or bodily fluid comprises optic nerve cells. 28.The method for diagnosing glaucoma of claim 22, wherein the cell orbodily fluid comprises retinal cells.
 29. The method for diagnosingglaucoma of claim 22, wherein the cell or bodily fluid comprises abodily fluid selected from the group consisting of glaucomatous cellextract, fluid from the anterior chamber of the eye, blood, lymph, andserum.
 30. The method for diagnosing glaucoma of claim 22, furthercomprising amplifying the complementary nucleic acid molecule obtainedfrom a sample using a nucleic acid amplification method.
 31. The methodfor diagnosing glaucoma of claim 22, wherein the nucleic acidamplification method is selected from the group consisting of polymerasechain amplification, ligase chain reaction, oligonucleotide ligationassay, thermal amplification, and transcription base amplification. 32.A method for prognosing glaucoma in a sample obtained from a cell or abodily fluid by detecting a polymorphism in a promoter region of theoptineurin gene, comprising the steps of: (A) incubating underconditions permitting nucleic acid hybridization, a marker nucleic acidmolecule, said marker nucleic acid molecule having a nucleic acidsequence that specifically hybridizes to a sequence selected from thegroup consisting of SEQ ID NO: 1 and complement thereof, and acomplementary nucleic acid molecule obtained from a sample, whereinnucleic acid hybridization between said marker nucleic acid molecule andsaid complementary nucleic acid molecule permits the detection of saidpolymorphism; (B) permitting hybridization between said marker nucleicacid molecule and said complementary nucleic acid molecule; and (C)detecting the presence of said polymorphism, wherein the detection ofsaid polymorphism is prognostic of glaucoma.
 33. The method forprognosing glaucoma of claim 32, wherein said polymorphism is a singlenucleotide polymorphism.
 34. The method for prognosing glaucoma of claim32, wherein said marker nucleic acid molecule has a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 3 through
 463. 35. Themethod for prognosing glaucoma of claim 32, further comprising a secondmarker nucleic acid molecule.
 36. A method for diagnosing or prognosingglaucoma in a sample obtained from a cell or a bodily fluid by detectinga polymorphism in a promoter region of the optineurin gene, comprisingthe steps of: (A) incubating under conditions permitting nucleic acidhybridization, a marker nucleic acid molecule, said marker nucleic acidmolecule having a nucleic acid sequence that specifically hybridizes toa optineurin promoter sequence or its complement, and a complementarynucleic acid molecule obtained from a sample, wherein nucleic acidhybridization between said marker nucleic acid molecule and saidcomplementary nucleic acid molecule permits the detection of saidpolymorphism; (B) permitting hybridization between said marker nucleicacid molecule and said complementary nucleic acid molecule; and (C)detecting the presence of said polymorphism, wherein the detection ofsaid polymorphism is diagnostic or prognostic of glaucoma.
 37. Themethod for diagnosing or prognosing glaucoma of claim 36, wherein saidoptineurin promoter sequence comprises SEQ ID NO: 1 or a fragmentthereof.
 38. The method for diagnosing or prognosing glaucoma of claim36, wherein said marker nucleic acid is capable of specificallydetecting a single nucleotide polymorphism.
 39. The method fordiagnosing or prognosing glaucoma of claim 36, wherein said markernucleic acid molecule has a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 3 through
 463. 40. The method for diagnosingor prognosing glaucoma of claim 36, further comprising a second markernucleic acid molecule.
 41. A method for detecting the presence orabsence of a SNP sequence variation in a sample containing DNA,comprising contacting a labeled nucleic acid capable of detecting asingle nucleotide polymorphism selected from table 1 with the DNA of thesample under hybridization conditions and determining the presence ofhybrid nucleic acid molecules comprising the labeled nucleic acid. 42.The method of claim 41, wherein the sample containing DNA is derivedfrom a human with elevated intraocular pressure.
 43. The method of claim41, wherein the sample containing DNA is derived from a human withoutelevated intraocular pressure.
 44. A method for detecting the presenceor absence of an optineurin promoter sequence variation in a samplecontaining DNA, comprising providing amplification reaction primers thatdirect amplification of a selected nucleic acid region containing saidsequence variation within said optineurin promoter, amplifyng thenucleic acid defined by the amplification reaction primers, anddetermining the presence or absence of said sequence variation.
 45. Themethod of claim 44, wherein the determining the presence or absence ofsaid sequence variation comprises sequencing the amplified nucleic acid.46. The method of claim 44, wherein the determining the presence orabsence of said sequence variation comprises a hybridization assay. 47.A method for determining the presence of increased susceptibility to aglaucoma, or to a progressive ocular hypertensive disorder resulting inloss of visual field in a patient, or the severity or progression ofglaucoma in a patient, comprising providing amplification reactionprimers that direct amplification of a selected nucleic acid regioncontaining said sequence variation within said optineurin promoter,amplifying the nucleic acid defined by the amplification reactionprimers, and determining the presence or absence of said sequencevariation.
 48. A method for detecting a polymorphism comprising:obtaining a sample containing human genomic DNA, providing a nucleicacid molecule capable of detecting a single nucleotide polymorphismlocated with an optineurin promoter, and detecting the presence orabsence of said polymorphism.
 49. The method detecting a polymorphismaccording to claim 48, wherein said polymorphism is selected fromtable
 1. 50. A kit for determining the presence of increasedsusceptibility to a glaucoma, or to a progressive ocular hypertensivedisorder resulting in loss of visual field, or the severity orprogression of glaucoma in a patient, comprising a labeled nucleic acidcapable of detecting a single nucleotide polymorphism selected fromtable 1 and a means for detecting hybridization with the labeled nucleicacid, and instructions for using said kit.
 51. A kit for determining thepresence of increased susceptibility to a glaucoma, or to a progressiveocular hypertensive disorder resulting in loss of visual field in apatient, or the severity or progression of glaucoma in a patient,comprising amplification reaction primers that direct amplification of aselected nucleic acid region containing a characteristic nucleotide ofan optineurin promoter SNP sequence variant and an enzyme for amplifyingthe region containing said characteristic nucleotide.